Attenuated piscirickettsia salmonis bacterium

ABSTRACT

The present invention relates to an attenuated Piscirickettsia salmonis bacterium. The bacterium comprises mutations in the amino acid sequence of each of the rpoD, FecR, ATP-grasp domain protein, and FtsH gene products. The invention also relates to vaccines comprising the attenuated Piscirickettsia salmonis bacterium that are useful for the prevention of microbial pathogenesis. In addition, the invention relates to methods for the preparation of attenuated Piscirickettsia salmonis bacteria, and vaccines comprising such bacteria.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage, 35 U.S.C. § 371, of theinternational application PCT/EP2016/061862, filed May 25, 2016, whichclaims priority from GB1509004.6 filed May 26, 2015, the entiredisclosures of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

In the broadest aspect, the present invention relates to an attenuatedbacterium, its preparation, and its use in a live attenuated vaccine.

BACKGROUND

Salmon Rickettsial Syndrome, “SRS”, (also known as, Piscirickettsiosis,Coho salmon septicaemia, or Huito disease) is considered to be one ofthe most important disease problems facing the salmon farming industry.The bacterium Piscirickettsia salmonis is the causative agent of SRS.

SRS continues to evolve and new outbreaks continually occur which areincreasingly insidious and refractory to treatments. New outbreaksfrequently show increased bacterial virulence, clinical and pathologicalseverity and variable presentation under similar conditions of species,age and management measures.

SRS has proven very difficult to control. The use of antibiotics, bothprophylactically and during early infection, may inhibit the growth ofthe pathogen, but failure of antibiotic treatment is common, andantibiotic treatments have been largely unsuccessful in stopping diseaseoutbreaks.

Thus, there is a need for improved methods of controlling P. salmonis.

Vaccines based on live but attenuated micro-organisms (live attenuatedvaccines) induce a highly effective type of immune response. Generally,such vaccines induce stronger and more durable immunity than vaccinesbased on an inactivated pathogen as they activate all phases of theimmune system. Specifically, once an animal host has been vaccinatedwith a live attenuated vaccine, entry of the microbial pathogen into thehost induces an accelerated recall of earlier, cell-mediated and/orhumoral immunity which is able to control the further growth of theorganism before the infection can assume clinically significantproportions. Inactivated vaccines (based on killed micro-organisms orfragments of micro-organisms) are less likely to be able to achieve thesame magnitude and rapidity of response.

There is thus a need for an attenuated strain of P. salmonis, suitablefor use in a vaccine. The attenuated vaccine should substantially retainthe antigenic capacity of the wild-type strain in order to cause arobust immune response in the host, and thereby provide strong immunity.The vaccine should also be sufficiently avirulent to minimiseundesirable pathological effects. In addition, the live attenuatedvaccine strain should have substantially no likelihood of reversion to avirulent form.

The general approach for attenuating bacteria is the removal of one ormore virulence factors. In most cases however, virulence factors arerequired in order to induce immunity, and deletion of virulence factorsunavoidably impairs the immunogenic capacities of the bacterium.

It has now surprisingly been found that by mutating the rpoD, FecR,ATP-grasp domain protein, and FtsH gene products, an attenuated P.salmonis bacterium can be produced, without impairing the viability orimmunogenicity of such bacteria in vivo. By mutating a number of genesin parallel, the likelihood of reversion to a virulent form isminimised. Moreover, these genes were not previously known to relate tovirulence factors, and it is therefore surprising that they have nowbeen found to affect bacterial pathogenicity. This also offers thefurther advantage that the attenuated bacterium demonstratessubstantially the same level of immunogenicity as wild-type strains. Thedisclosed bacterium has therefore surprisingly been found to beextremely suitable for use in the preparation of live attenuatedvaccines.

SUMMARY

According to a first aspect, an attenuated Piscirickettsia salmonisbacterium is provided. The bacterium comprises a mutation in the aminoacid sequence of each of the rpoD, FecR, ATP-grasp domain protein, andFtsH gene products.

The attenuated bacterium has a reduced virulence relative to wild-typeP. salmonis. The attenuated bacterium is preferably avirulent and doesnot induce any symptoms of Salmon Rickettsial Syndrome when administeredto fish.

The attenuated bacterium preferably does not revert to a virulent strainafter serial passage in fish. For example, the attenuated bacteriumpreferably does not revert to a virulent strain after 2, 3, 4, 5, or 6passages in fish.

The attenuated bacterium is preferably capable of inducing immunologicalprotection against Salmon Rickettsial Syndrome when administered tofish. Indeed, the attenuated bacterium preferably provides protection tofish against SRS following subsequent challenge with a virulent strainof P. salmonis. Preferably, when measured in terms of accumulatedmortality, the attenuated bacterium provides more than 40%, more than50%, more than 60%, or more than 80% protection against SRS. Preferably,when measured in terms of accumulated mortality, the attenuatedbacterium provides 100% protection against SRS.

The mutations in each of the rpoD, FecR, ATP-grasp domain protein, andFtsH genes of the attenuated bacterium, which underlie the mutations inthe corresponding gene products, may be non-reverting mutations.

The mutations in the amino acid sequence of the rpoD, FecR, ATP-graspdomain protein, and FtsH gene products may be mutations relative to thesequence of the corresponding LF-89 wild-type protein, as derived fromthe LF-89 genomic sequence that is available under the GenBank accessionno. AMFF00000000.2, and as provided as Seq. ID No.s 17, 26, 40, and 54,respectively.

The attenuated bacterium may comprise at least one mutation in 1, 2, 3,or all 4, of the following regions:

-   -   a) amino acid residues 462-504 of the rpoD gene product,        provided as Seq. ID No. 17;    -   b) amino acid residues 39-137 of the FecR gene product, provided        as Seq. ID No. 26;    -   c) amino acid residues 118-251 of the ATP-grasp domain protein        gene product, provided as Seq. ID No. 40; and/or,    -   d) amino acid residues 152-274 of the FtsH gene product,        provided as Seq. ID No. 54.

The attenuated bacterium may comprise 1, 2, 3, or all 4, of thefollowing specific mutations:

-   -   a) an arginine to cysteine mutation at position 473 of the rpoD        gene product, provided as Seq. ID No. 17;    -   b) a premature stop codon at the position corresponding to        residue 83 of the FecR gene product, provided as Seq. ID No. 26;    -   c) a serine to proline mutation at position 184 of the ATP-grasp        domain protein gene product, provided as Seq. ID No. 40; and/or,    -   d) a methionine to isoleucine mutation at position 191 of the        FtsH gene product, provided as Seq. ID No. 54.

The amino acid residue numbers given throughout are defined on the basisof the sequences of the corresponding LF-89 wild-type proteins, as shownin Example 2 (and given as Seq. ID No.s 17, 26, 40, 54), and derivedfrom the LF-89 genomic sequence that is available under the GenBankaccession no. AMFF00000000.2.

The attenuated bacterium may be the strain PHARMAQ 001 deposited withthe European Collection of Cell Cultures, Public health England, CultureCollections, Porton Down, Salisbury SP4 OJG, United Kingdom, on 9 Oct.2014 with accession number 14100901.

According to a second aspect, the invention provides a live, attenuatedvaccine composition comprising:

-   -   (a) an attenuated Piscirickettsia salmonis bacterium of the        first aspect; and    -   (b) a pharmaceutically acceptable carrier or diluent.

The live, attenuated vaccine composition may be in freeze-dried form.

According to a third aspect, the invention provides a method ofproducing an attenuated bacterium in accordance with the first aspect.The method comprises:

-   -   1) subjecting an initial population of P. salmonis bacteria to        attenuating conditions to produce a putatively attenuated        bacterial population;    -   2) identifying clones of the putatively attenuated bacterial        population that have mutations in the amino acid sequences of        all of the rpoD, FecR, ATP-grasp domain protein, and FtsH gene        products; and then,    -   3) identifying and selecting clones that have mutations in the        amino acid sequence of all of the rpoD, FecR, ATP-grasp domain        protein, and FtsH gene products and that also exhibit reduced        virulence relative to wild-type bacteria of the genus        Piscirickettsia.

According to a fourth aspect, the invention provides a method of raisingan immune response in a fish. The method comprises administering to thefish an attenuated Piscirickettsia salmonis bacterium of the firstaspect.

According to a fifth aspect, the invention provides a method ofvaccinating a fish against Salmon Rickettsial Syndrome. The methodcomprises administering to a fish an immunologically-effective amount ofa vaccine composition, said vaccine composition comprising an attenuatedPiscirickettsia salmonis bacterium of the first aspect.

According to a sixth aspect, the invention provides an attenuatedPiscirickettsia salmonis bacterium of the first aspect, for use in amethod of vaccinating a fish.

According to a seventh aspect, the invention provides an attenuatedPiscirickettsia salmonis bacterium of the first aspect, for use in amethod of vaccinating a fish against Salmon Rickettsial Syndrome.

According to an eighth aspect, the invention provides a method ofdistinguishing the PHARMAQ 001 strain of Piscirickettsia salmonis fromother strains such as wild-type strains. More specifically, theinvention provides a method of distinguishing between wild-type andmutant alleles of a Piscirickettsia salmonis single nucleotidepolymorphism (SNP) located at the position corresponding to:

-   -   residue number 1417 of Seq. ID No. 1 (in the rpoD gene);    -   residue number 247 of Seq. ID No. 4 (in the FecR gene);    -   residue number 550 of Seq. ID No. 7 (in the ATP-grasp domain        protein gene); or,    -   residue number 573 of Seq. ID No. 10 (in the FtsH gene).

The method comprises:

i) amplifying by PCR the region of the nucleotide sequence containingthe SNP;

ii) including in the PCR reaction mix a nucleic acid probe having asequence complementary to one allele of the SNP, the probe comprising adetectable marker; and

iii) analysing the PCR product for the presence of the marker, whereinthe presence of the marker is indicative of the presence of the allele.

The method may further comprise including in the PCR reaction mix afirst nucleic acid probe having a sequence complementary to thewild-type allele of the SNP, and a second nucleic acid probe having asequence complementary to the mutant allele of the SNP, the first andsecond probes comprising different detectable markers.

The probe may comprise a 10-40 nucleotide subsequence of:

-   -   Seq. ID No. 1, the subsequence including residue number 1417;    -   Seq. ID No. 4, the subsequence including residue number 247;    -   Seq. ID No. 7, the subsequence including residue number 550; or,    -   Seq. ID No. 10, the subsequence including residue number 573.

Specifically, the probe may comprise Seq. ID No. 68, 69, 72, 73, 76, or77.

A PCR primer pair may be used in the method to amplify a region of atleast 50 nucleotides in length of the subsequence of:

-   -   Seq. ID No. 1, the subsequence including residue number 1417;    -   Seq. ID No. 4, the subsequence including residue number 247;    -   Seq. ID No. 7, the subsequence including residue number 550; or,    -   Seq. ID No. 10, the subsequence including residue number 573.

Specifically, the PCR primer pair may comprise Seq. ID No. 67 and 70, 71and 74, or 75 and 78.

DETAILED DESCRIPTION

P. salmonis

SRS is caused by the gram-negative bacterium, Piscirickettsia salmonis.This was the first “rickettsia-like” bacterium to be recognized as apathogen of fish.

P. salmonis is generally non-motile, obligate intracellular bacterium,pleomorphic but predominately coccoid, and 0.5-1.5 μm in diameter. It iscurrently placed in the class Gammaproteobacteria; order Thiotrichales;and family Piscirickettsiacaea, and has a closer relationship to, e.g.,Legionella and Coxiella, than to members of the genera Rickettsia. Thebacterium replicates by binary fission within membrane bound cytoplasmicvacuoles in fish cell lines and in the cells of tissues throughoutinfected fish. The bacteria occur either singularly or in groups, givingthe vacuole the appearance of a morula. When P. salmonis is examined byelectron microscopy, the bacterium displays the typical protoplasmicstructure of a prokaryote and the cell wall of a gram-negativebacterium.

The genome of P. salmonis strain LF-89 has been sequenced and publishedon at least three separate occasions (see, for example, Eppinger et al.Genome Announc. November/December 2013 vol. 1 no. 6), and is availablevia DDBJ/EMBL/GenBank under the accession no.s AMGC00000000.1,AMFF00000000.2, and ASSK00000000.2. Unless otherwise indicated, theLF-89 sequence used in the present application is the sequence availableunder the accession no. AMFF00000000.2. Specifically, unless otherwiseindicated, for the purposes of genetic and protein sequence comparisonin particular, this LF-89 sequence that is available under the GenBankaccession no. AMFF00000000.2 is considered to represent the sequence ofwild-type P. salmonis, and references to the wild-type in this contextshould be interpreted accordingly. The genomes of P. salmonis strainsEM-90 (NCBI Reference Sequence: NZ_JRHP00000000.1), A1-15972 (NCBIReference Sequence: NZ_JRAV00000000.2), and B1-32597 (NCBI ReferenceSequence: NZ_JRAD00000000.2) have also been published.

Attenuated Bacterium

The attenuated P. salmonis bacterium of the invention is attenuated bymeans of a mutation in each of the rpoD, FecR, ATP-grasp domain protein,and FtsH genes. Specifically, the attenuated bacterium comprises geneticmutations which result in mutations in the amino acid sequence of eachof the rpoD, FecR, ATP-grasp domain protein, and FtsH gene products,relative to the sequence of the wild-type proteins.

For the purposes of the invention, the term “gene product” isspecifically considered to refer to the protein resulting from theexpression of a gene.

For the purposes of the invention, a “mutation” is considered to be anyalteration in the gene or protein sequence relative to the wild-typesequence. Genetic mutations that are of interest are those that resultin a mutation (i.e. alteration) in the resulting amino acid sequence ofthe gene product relative to the wild-type amino acid sequence. Each ofthe mutations in the rpoD, FecR, ATP-grasp domain protein, and FtsHgenes can be any type of mutation, including an insertion, a deletion, asubstitution, or any combination of these, provided that the mutationleads to a change in the amino acid sequence of each of the rpoD, FecR,ATP-grasp domain protein, and FtsH gene products, relative to thewild-type protein sequence.

A functional gene product is a protein having the functionalcharacteristics of the wild-type protein. A rpoD, FecR, ATP-grasp domainprotein, or FtsH gene product that is at least partially defective in atleast one of its functions is considered to be an attenuated geneproduct. Any mutation resulting in an attenuated gene product isconsidered to be an attenuating mutation. The mutations in the aminoacid sequence of each of the rpoD, FecR, ATP-grasp domain protein, andFtsH gene products, relative to the wild-type proteins, are preferablyattenuating mutations.

Attenuating mutations in the rpoD, FecR, ATP-grasp domain protein, andFtsH gene products may knock-out the function of the gene productpartially or completely. The partial or total functional knock-out maybe achieved, for example, by making a mutation that results in thesynthesis of non-functional or partially functional polypeptide. Forexample, the mutation in the amino acid sequence may comprise theinsertion of a stop codon, or may result in the incorporation of anamino acid that is physically or chemically dissimilar to the wild-typeresidue. Such mutations may result in the production of a truncatedprotein, a misfolded protein, or a chemically inactive protein, forexample.

The mutations in each of the rpoD, FecR, ATP-grasp domain protein, andFtsH genes, which result in mutated gene products, are preferablynon-reverting mutations. These are mutations that show essentially noreversion back to the wild-type when the bacterium is used as a vaccine.

The possibility of reversion of the bacterium to full virulence is alsoeliminated by the fact the bacterium contains attenuating mutations infour independent genes.

Attenuated Genes

The gene rpoD encodes RNA polymerase sigma factor, which is aninitiation factor involved in promoting the attachment of RNA polymeraseto specific transcription initiation sites. The rpoD gene product isbelieved to be involved in the regulation of essential housekeepinggenes. For the avoidance of doubt, the wild-type P. salmonis rpoD genesequence that is mutated in the present invention is given as Seq. IDNo. 1, and the gene can be identified using the PCR primers of Seq. IDNo.s 2 and 3. The amino acid sequence of the full-length wild-typeprotein is given in Seq. ID No.s 14-17. The attenuated bacterium of theinvention comprises a mutation in the amino acid sequence of the rpoDgene product, relative to the sequence of the wild-type protein, whichis exemplified by the LF-89 sequence, given as Seq. ID No. 17.

The gene FecR encodes an iron dicitrate transport regulator. The FecRgene product is believed to be involved in regulating a number of genesinvolved in the uptake of iron and citrate. For the avoidance of doubt,the wild-type P. salmonis FecR gene sequence that is mutated in thepresent invention is given as Seq. ID No. 4, and the gene can beidentified using the PCR primers of Seq. ID No.s 5 and 6. The amino acidsequence of the full-length wild-type protein is given in Seq. ID No.s26-29. The attenuated bacterium of the invention comprises a mutation inthe amino acid sequence of the FecR gene product, relative to thesequence of the wild-type protein, which is exemplified by the LF-89sequence, given as Seq. ID No. 26.

The ATP-grasp domain protein gene encodes a protein with similarity toan alpha-L-glutamate ligase-related protein found in Pseudomonas(GenBank accession No. AP 014655.1). For the avoidance of doubt, thewild-type P. salmonis ATP-grasp domain protein gene sequence that ismutated in the present invention is given as Seq. ID No. 7, and the genecan be identified using the PCR primers of Seq. ID No.s 8 and 9. Theamino acid sequence of the full-length wild-type protein is given inSeq. ID No.s 40-43. The attenuated bacterium of the invention comprisesa mutation in the amino acid sequence of the ATP-grasp domain proteingene product, relative to the sequence of the wild-type protein, whichis exemplified by the LF-89 sequence, given as Seq. ID No. 40.

The gene FtsH encodes an ATP-dependent zinc metalloprotease, which actsas a processive, ATP-dependent zinc metallopeptidase for bothcytoplasmic and membrane proteins. The FtsH gene product is alsobelieved to play a role in the quality control of integral membraneproteins. For the avoidance of doubt, the wild-type P. salmonis FtsHgene sequence that is mutated in the present invention is given as Seq.ID No. 10, and the gene can be identified using the PCR primers of Seq.ID No.s 11 and 12. The amino acid sequence of the full-length wild-typeprotein is given in Seq. ID No.s 54-57. The attenuated bacterium of theinvention comprises a mutation in the amino acid sequence of the FtsHgene product, relative to the sequence of the wild-type protein, whichis exemplified by the LF-89 sequence, given as Seq. ID No. 54.

In some embodiments, one or more of the rpoD, FecR, ATP-grasp domainprotein, and FtsH gene products are entirely knocked-out, with theeffect that no functional protein is detectable. Thus, the mutation inthe amino acid sequence of the gene product is that there is no aminoacid sequence.

In other embodiments, the mutation may comprise the introduction of astop codon.

In some embodiments, the genes may be expressed at wild-type levels, butmutated so that the gene products have a different amino acid sequenceto that found in wild-type strains. The genetic mutation may result in adeletion, an insertion, and/or a substitution of one or more amino acidsin the gene product. The genetic mutation may result in full-length orsubstantially full-length gene products, or truncated gene products. Themutation may be a point mutation, affecting just one amino acid, or mayaffect more than one amino acid residue, such as, for example, affecting2-20 residues, 3-15 residues, 4-12 residues, or 5-10 residues.

For example, in one embodiment of the invention, the rpoD gene ismutated resulting in the replacement of arginine with cysteine atposition 473 in the amino acid sequence of the gene product. As a resultof this mutation, the protein encoded by the mutated rpoD gene hasdifferent functional properties to those of the wild-type protein.

In one embodiment of the invention, the FecR gene is mutated resultingin the insertion of a premature stop codon, for example, in the positionof residue 83, and therefore the production of a truncated gene product,having different functional properties to those of the wild-typeprotein.

In one embodiment of the invention, the ATP-grasp domain protein gene ismutated resulting in the replacement of serine with proline at position184 in the amino acid sequence of the gene product. As a result, themutated ATP-grasp domain protein has different functional properties tothose of the wild-type protein.

In one embodiment of the invention, the FtsH gene is mutated resultingin the replacement of methionine with isoleucine at position 191 in theamino acid sequence of the gene product. As a result, the proteinencoded by the mutated FtsH gene has different functional properties tothose of the wild-type protein.

In some embodiments, two, three, or all four, of the specific pointmutations described above in the rpoD, FecR, ATP-grasp domain protein,and FtsH genes may be present in combination. For example, in oneembodiment, the attenuated bacterium comprises the specific pointmutations described above in three of the rpoD, FecR, ATP-grasp domainprotein, and FtsH genes, and the fourth gene has a different mutation tothat described above. In one embodiment, the attenuated bacteriumcomprises all four of the specific point mutations described above inthe rpoD, FecR, ATP-grasp domain protein, and FtsH genes.

The bacterium preferably contains only defined mutations, which arefully characterised. It is less preferred to use a bacterium which hasuncharacterised mutations in its genome as a vaccine because there wouldbe a risk that the uncharacterised mutations may confer properties onthe bacterium that cause undesirable side-effects.

Production of Attenuated P. salmonis

In another aspect of the present invention, the invention providesmethods for identifying and/or producing attenuated P. salmonis clones.

The methods according to this aspect of the invention include subjectingan initial population of P. salmonis bacteria to attenuating conditions,thereby producing a putatively attenuated bacterial population.

According to this aspect of the invention, the “initial population of P.salmonis bacteria” can be any quantity of P. salmonis bacteria. Thebacteria, in certain embodiments are wild-type P. salmonis bacteria. Anumber of strains of P. salmonis have been isolated following outbreaksof SRS. Any of these isolated strains would potentially be suitable as astarting population for producing a putatively attenuated bacterialpopulation, including the following strains: AL 10 016, AL 10 008, AL 20218, AL 20 219, AL 20 223, AL 20 220, AL 20 470, AL 20 471, AL 20 455,AL 20 222, A1-15972, B1-32597, LF-89, EM-90. References to wild-type P.salmonis may refer to any of these strains. Preferably, however,references to wild-type P. salmonis refer to any of strains A1-15972,B1-32597, LF-89, or EM-90, such as in particular, strains A1-15972,LF-89, or EM-90. Unless otherwise indicated, however, for the specificpurposes of genetic and protein sequence comparison, the LF-89 sequencethat is available under the GenBank accession no. AMFF00000000.2 isconsidered to represent the sequence of wild-type P. salmonis, andreferences to the wild-type in this context should be interpretedaccordingly.

The bacteria used as a starting population for producing a putativelyattenuated bacterial population may alternatively contain one or moremutations relative to the wild-type, or other strain.

Preferably, the bacteria in the initial population are clonallyidentical or substantially clonally identical. In other words, thebacteria are preferably all derived from a single parental P. salmonisbacterial cell and/or have identical or substantially identicalgenotypic and/or phenotypic characteristics.

The term “attenuating conditions” refers to any condition or combinationof conditions which has or have the potential for introducing one ormore genetic changes (i.e., mutations) into the genome of a P. salmonisbacterium. Exemplary, non-limiting, attenuating conditions include, forexample, passaging bacteria in culture, transforming bacteria with agenome-insertable genetic element such as a transposon (e.g., atransposon that randomly inserts into the P. salmonis genome), exposingbacteria to one or more mutagens (e.g., chemical mutagens or ultravioletlight), and any other suitable methods.

Indeed, the attenuating mutations may be introduced by any suitablemethod. A possibility to introduce a mutation at a predetermined site,deliberately rather than randomly, is offered by recombinantDNA-technology. Such a mutation may be an insertion, a deletion, areplacement of one or more nucleotides, or any combination of these,with the only proviso that the genetic mutation leads to a mutation inthe amino acid sequence of the resulting gene product.

For example, one possible method includes cloning the DNA sequence ofthe wild-type gene into a vector, such as a plasmid, and inserting aselectable marker into the cloned DNA sequence or deleting a part of theDNA sequence, resulting in its inactivation. A deletion may beintroduced by, for example, cutting the DNA sequence using restrictionenzymes that cut at two points in or just outside the coding sequenceand ligating together the two ends in the remaining sequence. A plasmidcarrying the inactivated DNA sequence can be transformed into thebacterium by known techniques such as electroporation and conjugation.It is then possible by suitable selection to identify a mutant whereinthe inactivated DNA sequence has recombined into the chromosome of thebacterium and the wild-type DNA sequence has been renderednon-functional by homologous recombination.

In some embodiments, one or more further mutations may be introducedinto the bacteria to generate strains containing mutations in genes inaddition to those in the rpoD, FecR, ATP-grasp domain protein, and FtsHgenes.

When bacterial cells are attenuated by passaging in vitro, the cells maybe passaged any number of times, such as for example, at least 10, 20,40, 60, 80, 100, 120, or more times in vitro.

The initial population of P. salmonis, after being subjected toattenuating conditions, is referred to as a putatively attenuatedbacterial population. Individual clones of the putatively attenuatedbacterial population can be obtained by standard microbiologicaltechniques including, for example, serially diluting the cells andplating out individual cells on appropriate media.

Once obtained, the individual clones of the putatively attenuatedbacterial population are assayed for mutations in each of the rpoD,FecR, ATP-grasp domain protein, and FtsH genes. The mutated genesequences are then analysed to determine whether the resulting aminoacid sequences have any mutations. Mutations in the amino acid sequencesof the gene products are considered to be any differences in the aminoacid sequences compared to the wild-type P. salmonis sequence.

Any suitable method may be used to determine whether a putativelyattenuated P. salmonis bacterium exhibits mutations in each of the rpoD,FecR, ATP-grasp domain protein, and FtsH genes, and consequently anymutations in the amino acid sequence of any of the rpoD, FecR, ATP-graspdomain protein, and FtsH gene products.

One method by which mutations in these genes may be identified is byamplifying and sequencing portions of the genes. Any suitable PCR methodmay be used to amplify portions of the genes and pairs of PCR primerssuitable for amplifying specific portions of the rpoD, FecR, ATP-graspdomain protein, and FtsH genes are given in Seq. ID No.s: 2 and 3; 5 and6; 8 and 9; and 11 and 12, respectively. The amino acid sequence of thegene product can be determined from the genetic sequence using anysuitable computational tool.

For the avoidance of doubt, the primers given in Seq. ID No.s: 2 and 3;5 and 6; 8 and 9; and 11 and 12, may also be used to identify the genesreferred to as rpoD, FecR, ATP-grasp domain protein, and FtsHrespectively.

The portions of the genes amplified using the PCR primers of Seq. IDNo.s: 2 and 3; 5 and 6; 8 and 9; and 11 and 12 are regions of the genes,which when mutated, have been found to be particularly associated withthe attenuation of the bacteria. Specifically, differences between theamino acid sequences of these portions of the rpoD, FecR, ATP-graspdomain protein, and FtsH gene products, and the wild type sequences, maybe indicative of attenuating mutations in the gene products. Therefore,in some embodiments, references to mutations in the amino acid sequenceof the rpoD, FecR, ATP-grasp domain protein, and FtsH gene productspreferably refer to the presence of mutations in the amino acid sequenceof those portions of the gene products corresponding to the regions thatmay be amplified by the PCR primers of Seq. ID No.s: 2 and 3; 5 and 6; 8and 9; and 11 and 12.

Differences in the amino acid sequences of the rpoD, FecR, ATP-graspdomain protein, and FtsH gene products between the putatively attenuatedand wild-type P. salmonis bacteria may in some cases not be accompaniedby reduced function of the gene product. For the purposes of theinvention, such mutations are not considered to be attenuatingmutations. Non-attenuating mutations may be found in the portions of thegene products corresponding to the regions amplified using the PCRprimers of Seq. ID No.s: 2 and 3; 5 and 6; 8 and 9; and 11 and 12, orelsewhere in the genes.

In some embodiments, some (such as 1, 2, or 3) of the rpoD, FecR,ATP-grasp domain protein, and FtsH gene products may include attenuatingmutations, while the remainder of the rpoD, FecR, ATP-grasp domainprotein, and FtsH gene products contain non-attenuating mutations.

The clones that have been identified as having mutations in the aminoacid sequence of each of the rpoD, FecR, ATP-grasp domain protein, andFtsH gene products, relative to the wild-type sequence, are then testedfor virulence.

Individual clones that are identified as having mutations in the aminoacid sequence of each of the rpoD, FecR, ATP-grasp domain protein, andFtsH gene products can be tested for virulence by any suitable method.For example, the attenuated bacteria may be administered to an animalthat is susceptible to infection by the wild-type version of thebacterium, and the presence and severity of disease determined.

In the present context, “an animal that is susceptible to infection by awild-type P. salmonis bacterium” is an animal that shows at least oneclinical symptom after being challenged with a wild-type P. salmonisbacterium. Such symptoms are known to persons of ordinary skill in theart. For example, in the case of a putatively attenuated P. salmonisstrain that exhibits mutations in the amino acid sequence of each of therpoD, FecR, ATP-grasp domain protein, and FtsH gene products, the straincan be administered to, for example, salmon (which are normallysusceptible to infection by wild-type P. salmonis). Clinical symptoms ofSRS in salmon are known to the skilled person.

In some embodiments, the symptoms investigated may include theaccumulated mortality of a population. If the accumulated mortality islower in animals challenged with the putatively attenuated P. salmonisstrain, compared to fish that have been infected with a wild-type P.salmonis strain, then the putatively attenuated P. salmonis strain isdeemed to have reduced virulence.

In some embodiments, the symptoms investigated may include the presenceor accumulation or P. salmonis genomes in tissue samples taken fromanimals challenged with the putatively attenuated P. salmonis strain. Ifthe presence of P. salmonis DNA is reduced compared to fish that havebeen infected with a wild-type P. salmonis strain, then the putativelyattenuated P. salmonis strain is deemed to have reduced virulence.

Thus, if the putatively attenuated P. salmonis strain, when administeredto salmon, results in fewer and/or less severe symptoms when compared tofish that have been infected with a wild-type P. salmonis strain, thenthe putatively attenuated P. salmonis strain is deemed to have “reducedvirulence”. Any degree of reduction in any relevant symptoms willidentify the putatively attenuated strain as having reduced virulence.

For the purposes of the invention, any strain that is found to have areduced virulence is considered to be an attenuated strain. In preferredembodiments, the putatively attenuated strain is avirulent.

Clones that exhibit mutations in the amino acid sequence of each of therpoD, FecR, ATP-grasp domain protein, and FtsH gene products, and thatalso exhibit reduced virulence relative to wild-type P. salmonis areidentified as attenuated P. salmonis clones of the present invention.

An exemplary, live, attenuated P. salmonis clone of the presentinvention, which exhibits non-reverting genetic mutations resulting inmutations in the amino acid sequence of each of the rpoD, FecR,ATP-grasp domain protein, and FtsH gene products is the straindesignated PHARMAQ 001.

Specifically, relative to wild-type P. salmonis, PHARMAQ 001 has beenfound to have mutations located at positions corresponding to:

-   -   residue number 1417 of Seq. ID No. 1 (in the rpoD gene);    -   residue number 247 of Seq. ID No. 4 (in the FecR gene);    -   residue number 550 of Seq. ID No. 7 (in the ATP-grasp domain        protein gene); and,    -   residue number 573 of Seq. ID No. 10 (in the FtsH gene).

For the purpose of the present disclosure, the mutations found in the P.salmonis strain PHARMAQ 001 are considered to represent singlenucleotide polymorphisms (SNP). Various methods of distinguishingbetween SNP alleles are known to the skilled person, and can be used todetermine whether a given strain is PHARMAQ 001. In particular, variousdifferent methods have been developed for the detection of specificalleles, or DNA sequence variants, at the same locus by polymerase chainreaction. For example, suitable methods may be based on the use PCRprimers with a 3′ end specific for one of the allelic variants, or onthe use of nucleic acid probes having a sequence complementary to thesequence of one particular individual allelic variant.

PHARMAQ 001 has been deposited with the European Collection of CellCultures, Public health England, Culture Collections, Porton Down,Salisbury SP4 OJG, United Kingdom, on 9 Oct. 2014 and was assignedaccession number 14100901.

Vaccine

A vaccine comprising the disclosed attenuated bacterium may beformulated using known techniques.

It has now surprisingly been found that an attenuated P. salmonisbacterium having a combination of mutations in the amino acid sequencesof the rpoD, FecR, ATP-grasp domain protein, and FtsH gene productsgives a vaccine having superior properties for at least two reasons.

Firstly, due to the presence of multiple mutations in four independentgenes there is a significantly reduced chance of reversion ofattenuation of the bacterium. Therefore, the bacterium can survive inthe vaccinated host for a long time and at high levels, resulting inbetter protection.

Secondly, the disclosed bacterium does not cause reduced immunogenicitycompared to wild type strains because antigens important forimmunogenicity are still expressed.

The vaccine composition preferably comprises a live, attenuated P.salmonis bacterium and a pharmaceutically acceptable carrier.

Examples of pharmaceutically acceptable carriers or diluents useful inthe present invention include water, a preservative, culture medium,stabilisers such as SPGA, carbohydrates (e.g. sorbitol, mannitol,starch, sucrose, glucose, dextran), proteins such as albumin or casein,protein containing agents such as bovine serum or skimmed milk, andbuffers (e.g. phosphate buffer).

The vaccine may or may not comprise an adjuvant. Adjuvants arenon-specific stimulators of the immune system. They enhance the immuneresponse of the host to the vaccine. Examples of adjuvants known in theart are Freunds Complete and Incomplete adjuvant, vitamin E, non-ionicblock polymers, muramyldipeptides, ISCOMs (immune stimulatingcomplexes), Saponins, mineral oil, vegetable oil, and Carbopol.

Vaccine formulations comprising the disclosed attenuated bacterium canbe prepared in the form of a suspension or in a lyophilized form or,alternatively, in a frozen form. If the formulation is to be frozen,glycerol or other similar agents may be included in the formulation toenhance the stability of the bacterium when frozen.

Reconstitution is advantageously effected in a buffer at a suitable pHto ensure the viability of the bacteria.

The combined administration of several vaccines is desirable, in orderto save time, effort, and money. Preferably, vaccine formulationscomprising the disclosed attenuated bacterium may be used together withother vaccines, such as, for example, an inactivated, oil adjuvantedvaccine. The vaccines may be administered together, for example, in asingle composition, or separately.

Vaccinated Species

The animal to which the vaccine comprising the disclosed attenuatedbacterium is administered is preferably a fish. The vaccine may beadministered to any species of fish that is susceptible to SRSinfection.

Of particular note, the vaccine is suitable for treating fish of theorder Samoniformes. For example, the claimed formulation may be used totreat salmon such as Atlantic and Pacific salmon, such as Coho salmon,and trout such as rainbow trout and brown trout.

Method of Vaccination

The present invention includes methods of vaccinating fish against P.salmonis infection, such as SRS.

The methods according to this aspect of the invention compriseadministering to a fish an immunologically-effective amount of a vaccinecomposition comprising a live, attenuated P. salmonis bacterium of theinvention. The expression “immunologically-effective amount” means theamount of vaccine composition required to invoke the production ofprotective levels of immunity in a host upon vaccination.

The vaccine composition may be administered to the host in any mannerknown in the art. In particular, the vaccine formulation may be suitablefor parenteral administration, such as by intraperitoneal injection.

An infection caused by a microorganism, especially a pathogen, maytherefore be prevented by administering an effective dose of a vaccineprepared according to the invention.

The dosage of the vaccine employed will be dependent on various factorsincluding the size and weight of the host, the type of vaccineformulated, and the formulation.

For example, a dosage for Atlantic salmon, Coho salmon or rainbow trout,with an average weight of 25-30 grams, may comprise the administrationof from 1×10² to 1×10¹⁰, 1×10³ to 1×10⁹, or 1×10⁴ to 1×10⁸ TCID₅₀ perfish, such as from 1×10⁴ to 1×10⁷ TCID₅₀ per fish. As the skilled personwill appreciate, the preferred dosage may depend on the age, weight andtype of fish to be vaccinated, and the mode of administration. Forexample, dosages may need to be increased for larger, more robust fish,and decreased for smaller, more delicate fish.

EXAMPLES

The invention will now be explained in further detail in the followingExamples, which demonstrate the development of the claimed live,attenuated bacterium, and its use in a vaccine.

Example 1: Isolation of the PHARMAQ 001 Strain and Attenuation of theStrain

The P. salmonis strain used as the starting bacterial population for theproduction of an attenuated bacterium was a strain originally isolatedfrom an outbreak of SRS in Atlantic salmon in the X region in Chile.

The isolate was for the six first passages cultivated in the presence ofeukaryotic cells.

For the next passages until passage 104, the P. salmonis isolate wascultivated in cell free insect cell medium at 20° C. To secure ahomogenous culture, the passage 104 culture was serially diluted ininsect cell medium and seeded into 96 well cell culture plates. Bacteriagrown in chosen wells were further passed into new wells at an earlystage when it was most likely that the growth originated from a singlebacterium.

After a total of 111 passages, one clone from the wells was inoculatedinto a spinner flask and cultivated in insect cell medium. This passagewas used as the origin of the putatively attenuated bacterialpopulation, and the isolate was named “PHARMAQ 001”, which correspondsto passage 113.

The bacterial isolate was verified to be P. salmonis using a commercialkit “SRS Fluorotest Directo” from Bios Chile, Chile.

PHARMAQ 001 has been deposited with the European Collection of CellCultures, Public health England, Culture Collections, Porton Down,Salisbury SP4 OJG, United Kingdom, on 9 Oct. 2014 and was assignedaccession number 14100901.

Example 2: Analysis of PHARMAQ 001

The genomes of PHARMAQ 001 and the virulent starting strain weresequenced and the sequences were compared.

Genetic differences between the two strains, i.e. mutations accumulatedby the PHARMAQ 001 strain during its production, were identified.

Because these genetic differences underpin the observed differences inthe virulence of PHARMAQ 001 and the starting strain, mutations wereidentified in PHARMAQ 001 that resulted in a significant change in theamino acid sequence of the encoded protein. Specifically, foursignificant mutations in PHARMAQ 001 relative to the starting strainwere identified. The identity of the four genes was determined, and thegenes were found to be rpoD, ATP-grasp domain protein, and FtsH (allannotated by the IGS Prokaryotic Annotation Pipeline), and FecR (GenBankreference: KGB63484.1)

PCR primers were designed to allow the specific region of each of therpoD, ATP-grasp domain protein, FtsH, and FecR genes containing theidentified mutation to be amplified. In each of the gene sequences shownbelow, the mutation is shown in bold and underlined, and the primerbinding sites are highlighted in grey and underlined.

PHARMAQ 001 rpoD gene sequence (Seq. ID No. 1):

ATGGATCAACAAGAAAAAAGGTCGCAGTTTAAAGAACTCATTGTTCGAGGTAAACAGCAAGGCTTTTTAACGTTTACAGAGGTAAACGATCATCTTCCGGATGATATGAGCAGCCCGGAAGAAGTTGAAGAGATCGTTGCAATGATTAGCGACATGGGCATCCCCGTCTATGAAACTGCACCCGATCCTGACAGCTTACTCATGAATGAGCATGCCAGCTCTGCCGAAGATGATGCTGACGATGCCGTTGCAGCGCTCGATTCAGATGCTGAGTTTGGGCGAACAACCGACCCAGTACGCATGTATATGCGCGAGATGGGCAGCGTTGAGCTATTAACGCGCCAAGGTGAAATTGAGCTGGCTAAACGCATCGAGGAAGGCGTCAAACAAGCCTTTGAGGCAATCGCCCATTACCCACAAAGCACAGCGATTATTCTTGAAGAATATGCAAGATTTGAAGCCGAAGAAATCCGTTTAGATGATATTATCAGTGGCTATATCACCGAAGAAGATGAAGCTCCGACAAGCAACATCGGCTCCATGCTTGATGATGCCAATAAAGCCGATGATAATTTTGAAGCCGCCTTGACAGAAGACGACAGTACTGATGACGGTGAGGGTGAGGATGACAATGAAGAAATCCCCGTCGATAACACATTGGATGTTGAGGAAGCGGCAGAGCGTTTTGCCGAGCTAAAAGCTGCCTATGATGCGGTTATACAGGTTCAGGAAAAACACGGAATTCATCATAAAAAAACACAACAGCGTTGTGAAGAACTGTCTAAAGTATTAATGACATTTCGCCTAAAGCCCAATATGATCGATAAAATCACCAACTACTTACATGGCTTACTCAGCCAAGTCCGCAAACATGAGCGTCACATCATGGCTTTGTGCATTAATCAAGCGAAAATGCCCCGCAAGCTATTTATTGATATTTTCCCAGGCAATGAAACCAATCTAGAGTGGATAGAGTATCAAATTAAAGCCGAGCAATCTTACTCTGAAGCACTACAGTCCCTGGCTCCAGAAGTCACTCGTGCACAGAAAAAGCTCATCTCTCTTGAACAAGAATCAAACTTTGATGTCACTGCAATTAAAGAAGTCAATCGTAATATTTCTATTGGTGAGGCCAAGGCCCATCGTGCTAAAAAAGAAATGGTCGAAGCTAACTTGCGTCTGGTCATCTCCATTGCAAAAAAATACACCAATCGAGGCTTACAATTTCTCGACCTCATTCAAGAAGGCAATATTGGCCTAATGAAAGCGGTAGATAAATTCGAATACCGCCGTGGTTATAAATTCTCAACGTATGCAACATGGTGGATTCGCCAGGCAATTACCCGCTCAATTGCTGATCAAGCGCGGACAATCCGGATTCCTGTACATATGATAGAGACAATTAACAAGCTTAAC T GCGTCTCACGTCAAATGATCCAAGAACTCGGCCGTGAAGCGACTCCTGAGGAGCTTTCTGAACGCATGGAGATGCCAGAGCATAAAATCCGTAAAATCCTCAAAATCGCTAAAGAGCCAATTTCTATGGAAACACCGATTGGCGATGACGAAGATTCACACTTAGGTGATTTTATTGAAGATACCACCATGCAACTCCCCGTTGACTCAACCATGGGTGATGCGTTAAAGCAAGCCACCAGTGATATTCTCGAAAACCTCACGCCACGTGAAGCAAAAGTCCTTAGAATGCGCTTTGGTATCGATATGAATACTGACCATACGCTAGAGGAAGTTGGCAAACAATTTGACGTAACTCGCGAGCGTATTCGCCAAATTGAAGCCAAAGCCTTACGTAAACTGCGCCACCCAACTCGCTCAGAAATTTTGAAGAGTTTTCTTGACTC AGAAGAATAA

PCR Primers:

Forward rpoD (Seq. ID No. 2): CCAGGCAATTACCCGCTCAAReverse rpoD (Seq. ID No. 3): TCGCCAATCGGTGTTTCCAT

PHARMAQ 001 FecR gene sequence (Seq. ID No. 4):

ATGAAAATTAATCATCAGCCTGGCGGCATAATGTTGATAATGAATAATCATGGTGAAGTGATGACAAGCTACTCACATATGATGATTTTTTTTtCAAATTATGGCGAAAAAGTAAAGATTGAGAATCAGCGTATCTTAAATGATAATAAACTGCTATTTAGTAATAGAGTGAGTAGGGTGCGTTATCGGCCTTGTCTTATTATTGATGCAAAAGATGCTTTATCAGTCTGTTCTGGCGTGTTTCA T TAAGTAAAAAATGAATTCGGAGTTGTTGTTGCAAGCTCTCTTAATGTGATGATATACGATTATAAATCAATGTCAGATGAGGATATTATTCATATTTTAAAGTCTGTCAAAAAACATCCAAAATTATCTTTAATAGAAAGCAAGATACTTTTTTTAAAAGTGGTAATGAAAGGGATAAAATGTCGCCATATTGAGTCGTTACTTAAGGTATCCGGAAGTACTGTGTATACGTATTGTATGAATATCAAGTCAAAAGCAAATATTTTtAGTTTTAAAGGGCAGTCCGTAATTCAGGAATTAGAAAAAAGTCAATTTTTTAGTGATATTTCTATAAAGATAAATATAGATTTCTATAACATTAATAATGGAGTAAATGAGAAAAATGTTTGCC AAGTATATAGCCTAGCTTAA

Primers:

Forward FecR (Seq. ID No. 5): TGGTGAAGTGATGACAAGCTACReverse FecR (Seq. ID No. 6): ACACAGTACTTCCGGATACCTT

PHARMAQ 001 ATP-grasp domain protein gene sequence (Seq. ID No. 7):

ATGATCAGCCTGTGGAAGACCTATCAGGCGCTTAAAACAAAGGGCATTTTAGGCATTAATCAGCGTAATGCTGACTTTATTATTCGCTATAATCAGCGCAAATACTACCCTTTAGTCGATGATAAAATCATGACAAAAACCCTTGCGATTAAAGATGGTATTGCCGTCCCTAAATTATATGCAACCCTTAAAACTGACCATGATACTCACCATCTGGAGCAAATTTTAGCCAATCGAACGGATTTTGTCATTAAACCGGCCCGTGGTGCTGGCGGTGATGGTATTTTAGTCATTACCAACCGCCATGGTGAGCGTTTTCGCAAAGTCAGTGGTGCACTGTTACACTTAGACGATATTCGTCATCACATTTCTAATATCTTAAGTGGGGTATACAGTCTTGGTGGCCAACGTGATCAGGCCATGATTGAATACCGCGTACAATTTGATCCATTATTTAAAAAAATCAGCTATCAAGGTGTGCCCGATATTCGTATTATTGTCCTAAAAGGCTATCCTGCGATGGCGATGGTGCGTCTACC CACTCGGCTC CCTGATGGCAAAGCCAACCTTCATCAAGGTGCAATTGGCGTTGGCATTGACTTAACAACAGGCATCACCTTAGAAGGTGTTTGGATGAATGACCCAATTCATGAACATCCGGATACTGGCTATGCTGTACCAGGCTTACAGATTCCTCACTGGGATCAtTTTTTAAACCTTGCTGCACGCTGTTACGAGCTTACTCAACTAGGTTATTTAGGTGTGGATATTATCCTTGATAAAGACAAAGGCCCACTCATGCTTGAGCTTAATGCGCGTCCTGGTTTAAATATTCAAATTGCGAATAATAGCGGCTTATTGCATCGATTACGTTTCATTGAGCAACAAAATCAACAACGCACAGCCGATGAACGCATTGCTTTCATCAAAC ATCAGTTCGCAAAAATATAA

Primers:

Forward ATP-grasp domain protein (Seq. ID No. 8): GGTGAGCGTTTTCGCAAAGTReverse ATP-grasp domain protein (Seq. ID No. 9): TCAAGCATGAGTGGGCCTTT

PHARMAQ 001 FtsH gene sequence (Seq. ID No. 10):

ATGATTAAAAACATCATGCTATGGCTGGTCATTGCTTTGGTGTTGGTGACTGTGTTTAGTAATTTAGGCCCACGTCAGCAGTCGGTGAATCGGCTAGATTATTCAACATTTGTTAAAGACATCAATAATGGTCAAGTAAAAAGCGTTATCATTGATGGTTTGAATATTAAAGGACAAACCTCAAGTGGGACGCCATTTGCTACTTATATTCCGTGGAAAGATCCATTTTTAATGGATCAGATGCTGGCGAAAAATGTCACAATTGCTGCTAAACCACCTGAGCAGCGGAGCTGGTTATTGTCTGCATTAATCAGTTGGTTCCCTGGTATTTTATTAATTGCGATTTGGATTTTCTTCTTGCGGCAGATGCAAGGCGGTGGTGGTGGTAAGGGCATGATGTCCTTTGGTTCCAGTAAGGCACGTCTGCTTGGTGAAGATCAAATTAAAGTTAACTTTGCTGATGTTGCTGGCTGTGAAGAGGCTAAAGAAGAAGTAAAAGAACTGGTCGATTTTCTGCGTGACCCAACCAAATTCCAAAAGTTAGGCGGCAAAATTCCGCAAGGGGTATTGAT A GTTGGCCCACCTGGAACAGGTAAGACGCTATTAGCTAAAGCCATTGCAGGTGAGGCGAAAGTCCCGTTCTTTTCTATTTCAGGCTCTGATTTTGTTGAAATGTTCGTCGGTGTCGGTGCATCGCGGGTGCGTGATATGTTTGATCAGGCAAAAAAACGTGCACCGTGTATTATCTTTATCGATGAGATTGATGCAGTGGGCCGTCACCGTGGCTCAGGTATGGGCGGTGGTCATGACGAACGTGAGCAGACCTTAAATCAAATGCTGGTCGAGATGGATGGTTTTGAGGGAACCGAAGGGGTGATTGTCATTGCCGCGACGAATCGTCCGGATGTATTGGACCCGGCATTATTGCGTCCCGGGCGTTTTGATCGCCAGGTCAGTGTCGGGCTTCCCGATGTCAAAGGCCGTGAGCAGATTCTAAAAGTGCATATGCGTAAGGTGCCTTTGGGAGATGATGTTAAAGCGTCATTGATCGCCCGTGGTACGCCTGGGTTCTCAGGAGCGGATTTGGCGAACTTGGTCAATGAAGCCGCACTCTTTGCCGCGCGTAAAGATAAAACCGTGGTTGCTATGCGTGAGTTTGATGATGCCAAAGATAAAATTTTGATGGGCACTGAGCGCCGTTCGATGGCAATGACCGAAGAGCAAAAACGTTTAACCGCCTTTCATGAGGCAGGGCACGCGATTGTCGGGTGTTTGGTACCTGATCATGATCCGGTGTATAAAGTCTCGATTGTGCCGCGGGGTCGTGCCTTAGGTGTGACCATGTATCTGCCTGAAGAGGATAGTTATGGTTATTCACGCGAGCGCTTGGAGAGCTTAATTTCGAGTATGTATGGCGGACGTATTGCTGAAGCTTTAGTCTTTGGTGTTGAGAAAGTAACGACTGGGGCATCGAATGACATTGAAAAAGCCTCAGAAGTGGCGCGCAATATGGTGACAAAGTGGGGGCTGTCTGAGCGCTTAGGGCCGATATTATATGGACAAGAAGGCGGTGATCCGTTTGGTTATGGTGCGGGTAAAGGCACGCCGGAATTTTCAGATCAAACCTCTGTTGCTATTGATGAGGAAGTACGTCAGATCATTGATCGTAATTATACACGCGCTGAGAGCATTCTAATCAATAATCGGGATATTCTTGATGCGATGGCGGATGCGTTGATGGTCTATGAGACGATTGATCGTGACCAAGTGGCTGATCTAATGGCGCGTCGGCCGGTGAAAGCACCGAAAGATTGGGATCAGCCCTCTGATGAGAGTGGCTCATCAGCATCTGGTGATGAGTTACAACCTCTTGATGCTAATATCAATACTGATATTAATGAGACTAAGAGCGCTGATCAAGAGACAGATCAGGGCGCGCCGTCACCAGAAATAAAGGGTAAACCAGCGGATGATCCTACCTAA

Primers:

Forward FtsH (Seq. ID No. 11): TGGTTCCAGTAAGGCACGTCReverse FtsH (Seq. ID No. 12): ACAATCACCCCTTCGGTTCC

The mutations in the rpoD, FecR, ATP-grasp domain protein, and FtsHgenes observed in PHARMAQ 001 have been found to be unique by comparisonwith DNA sequences from other strains of P. salmonis. For the purpose ofthe present disclosure, the mutations found in the P. salmonis strainPHARMAQ 001 are considered to represent single nucleotide polymorphismsand are located at positions corresponding to:

-   -   residue number 1417 of Seq. ID No. 1 (in the rpoD gene);    -   residue number 247 of Seq. ID No. 4 (in the FecR gene);    -   residue number 550 of Seq. ID No. 7 (in the ATP-grasp domain        protein gene); and,    -   residue number 573 of Seq. ID No. 10 (in the FtsH gene).

Table 1 shows the occurrence of the specific mutations in the rpoD,FecR, ATP-grasp domain protein, and FtsH genes observed in PHARMAQ 001(described in Example 2) in to different wild-type virulent strains ofP. salmonis.

TABLE 1 Gene Strain Origin Group rpoD FecR ATP-gbp FtsH PHARMAQ 001Atlantic salmon, Chile EM-90 like X X X X AL 10 005 Atlantic salmon,Chile EM-90 like n n n n AL 20 218 Atlantic salmon, Chile EM-90 like n nn n AL 20 223 Atlantic salmon, Chile LF-89 like n n n n AL 20 220 Trout,Chile LF-89 like n n n n AL 20 471 Trout, Chile LF-89 like n n n n AL 20222 Trout, Chile LF-89 like n n n n A1-15972 Atlantic salmon, ChileEM-90 like n n n n B1-32597 Coho salmon, Chile LF-89 like n n n n LF-89Coho salmon, Chile LF-89 n n n n EM-90 Atlantic salmon, Chile EM-90 n nn n

In Table 1, “X” indicates that the specific mutation found in PHARMAQ001 (described in Example 2) in each of the rpoD, ATP-grasp domainprotein, FtsH, or FecR genes is present, and “n” indicates that themutation is not present. As shown in Table 1, none of the strains of P.salmonis that were examined were found to possess any of the mutationsdescribed in Example 2 in the rpoD, FecR, ATP-grasp domain protein, andFtsH genes.

Thus, only PHARMAQ 001, and no other strains of P. salmonis, possessesthe described mutations in the rpoD, FecR, ATP-grasp domain protein, andFtsH genes.

The presence of these mutations in the rpoD, FecR, ATP-grasp domainprotein, and FtsH genes therefore provides a means of differentiatingand distinguishing the PHARMAQ 001 strain from other Piscirickettsiasalmonis strains.

A method of identifying the PHARMAQ 001 strain involves analysing theDNA sequence of the specific portions of each of the rpoD, FecR,ATP-grasp domain protein, and FtsH genes containing the identifiedmutation. The specific portions of the genes may be amplified bypolymerase chain reaction (PCR) using specific DNA primers (shown above)followed by DNA sequencing using standard methods. When compared to thesequences of LF-89 (and other wild type strains) the sequences of eachof the specific amplified portions of the rpoD, FecR, ATP-grasp domainprotein, and FtsH genes harbors a DNA point mutation which is specificand unique for PHARMAQ 001.

Mutations in the rpoD, FecR, ATP-grasp domain protein, and FtsH geneswere identified due to the fact that they are the four mutations inPHARMAQ 001 which result in a significant change in the amino acidsequence of a protein.

Amino acid sequence alignments between a number of virulent wild-typestrains of P. salmonis and the PHARMAQ 001 attenuated strain for each ofthe rpoD, FecR, ATP-grasp domain protein, and FtsH gene products wereinvestigated and are shown below.

The amino acid sequences of P. salmonis strains LF-89, EM-90, A1-15972,and B1-32597 were obtained from the published genome sequences of thesestrains (LF-89, DDBJ/EMBL/GenBank accession no. AMFF00000000.2; EM-90,GenBank accession no.: JRHP00000000.1; A1-15972, GenBank accession no.:JRAV00000000.2; and B1-32597, GenBank accession no.: JRAD00000000.2).The amino acid sequences for the other strains listed below wereobtained from the relevant virulent wild-type strain by means ofstandard PCR and sequencing methods using the PCR primer pairs describedabove (Seq. ID No.s 2 and 3, 5 and 6, 8 and 9, 11 and 12).

rpoD amino acid sequence alignment:

PHARMAQ_001MDQQEKRSQF KELIVRGKQQ GFLTFTEVND HLPDDMSSPE EVEEIVAMIS DMGIPVYETAA1-15972MDQQEKRSQF KELIVRGKQQ GFLTFTEVND HLPDDMSSPE EVEEIVAMIS DMGIPVYETAB1-32597MDQQEKKSQF KELIVRGKQQ GFLTFTEVND HLPDDMSSPE EVEEIVAMIS DMGIPVYETA EM-90MDQQEKRSQF KELIVRGKQQ GFLTFTEVND HLPDDMSSPE EVEEIVAMIS DMGIPVYETA LF-89MDQQEKRSQF KELIVRGKQQ GFLTFTEVND HLPDDMSSPE EVEEIVAMIS DMGIPVYETAAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20222---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20277---------- ---------- ---------- ---------- ---------- ---------- 61PHARMAQ_001PDPDSLLMNE HASSAEDDAD DAVAALDSDA EFGRTTDPVR MYMREMGSVE LLTRQGEIELA1-15972PDPDSLLMNE HASSAEDDAD DAVAALDSDA EFGRTTDPVR MYMREMGSVE LLTRQGEIELB1-32597PDPDSLLMNE HASSAEDDAD DAVAALGSDA EFGRTTDPVR MYMREMGSVE LLTRQGEIEL EM-90PDPDSLLMNE HASSAEDDAD DAVAALDSDA EFGRTTDPVR MYMREMGSVE LLTRQGEIEL LF-89PDPDSLLMNE HASSAEDDAD DAVAALDSDA EFGRTTDPVR MYMREMGSVE LLTRQGEIELAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20222---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20277---------- ---------- ---------- ---------- ---------- ---------- 121PHARMAQ_001AKRIEEGVKQ AFEAIAHYPQ STAIILEEYA RFEAEEIRLD DIISGYITEE DEAPTSNIGSA1-15972AKRIEEGVKQ AFEAIAHYPQ STAIILEEYA RFEAEEIRLD DIISGYITEE DEAPTSNIGSB1-32597AKRIEEGVKQ AFEAIAHYPQ STAIILEEYA RFEAEEIRLD DIISGYITEE DEAPTSNIGS EM-90AKRIEEGVKQ AFEAIAHYPQ STAIILEEYA RFEAEEIRLD DIISGYITEE DEAPTSNIGS LF-89AKRIEEGVKQ AFEAIAHYPQ STAIILEEYA RFEAEEIRLD DIISGYITEE DEAPTSNIGSAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20222---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20277---------- ---------- ---------- ---------- ---------- ---------- 181PHARMAQ_001MLDDANKADD NFEAALTEDD STDDGEGEDD NEEIPVDNTL DVEEAAERFA ELKAAYDAVIA1-15972MLDDANKADD NFEAALTEDD STDDGEGEDD NEEIPVDNTL DVEEAAERFA ELKAAYDAVIB1-32597MLHDANKADD NFEAALTEDD STDDAEDEGD NEEIPVDNTL DVEEAAERFA ELKAAYDAVI EM-90MLDDANKADD NFEAALTEDD STDDGEGEDD NEEIPVDNTL DVEEAAERFA ELKAAYDAVI LF-89MLDDANKADD NFEAALTEDD STDDGEGEDD NEEIPVDNTL DVEEAAERFA ELKAAYDAVIAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20222---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20277---------- ---------- ---------- ---------- ---------- ---------- 241PHARMAQ_001QVQEKHGIHH KKTQQRCEEL SKVLMTFRLK PNMIDKITNY LHGLLSQVRK HERHIMALCIA1-15972QVQEKHGIHH KKTQQRCEEL SKVLMTFRLK PNMIDKITNY LHGLLSQVRK HERHIMALCIB1-32597QVQEKHGIHH KKTQQRCEEL SKVLMTFRLK PNMIDKITNY LHDLLSQVRK HERHIMALCI EM-90QVQEKHGIHH KKTQQRCEEL SKVLMTFRLK PNMIDKITNY LHGLLSQVRK HERHIMALCI LF-89QVQEKHGIHH KKTQQRCEEL SKVLMTFRLK PNMIDKITNY LHGLLSQVRK HERHIMALCIAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20222---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20277---------- ---------- ---------- ---------- ---------- ---------- 301PHARMAQ_001NQAKMPRKLF IDIFPGNETN LEWIEYQIKA EQSYSEALQS LAPEVTRAQK KLISLEQESNA1-15972NQAKMPRKLF IDIFPGNETN LEWIEYQIKA EQSYSEALQS LAPEVTRAQK KLISLEQESNB1-32597NQAKMPRKLF IDIFPGNETN LDWIEYQIKA EQSYSEALQS LAPEVTRAQK KLISLEQESN EM-90NQAKMPRKLF IDIFPGNETN LEWIEYQIKA EQSYSEALQS LAPEVTRAQK KLISLEQESN LF-89NQAKMPRKLF IDIFPGNETN LEWIEYQIKA EQSYSEALQS LAPEVTRAQK KLISLEQESNAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20222---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20277---------- ---------- ---------- ---------- ---------- ---------- 361PHARMAQ_001FDVTAIKEVN RNISIGEAKA HRAKKEMVEA NLRLVISIAK KYTNRGLQFL DLIQEGNIGLA1-15972FDVTAIKEVN RNISIGEAKA HRAKKEMVEA NLRLVISIAK KYTNRGLQFL DLIQEGNIGLB1-32597FDVTAIKEVN RNISIGEAKA HRAKKEMVEA NLRLVISIAK KYTNRGLQFL DLIQEGNIGL EM-90FDVTAIKEVN RNISIGEAKA HRAKKEMVEA NLRLVISIAK KYTNRGLQFL DLIQEGNIGL LF-89FDVTAIKEVN RNISIGEAKA HRAKKEMVEA NLRLVISIAK KYTNRGLQFL DLIQEGNIGLAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20222---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20277---------- ---------- ---------- ---------- ---------- ---------- 421                                                         * PHARMAQ_001MKAVDKFEYR RGYKFSTYAT WWIRQAITRS IADQARTIRI PVHMIETINK LN C VSRQMIQA1-15972MKAVDKFEYR RGYKFSTYAT WWIRQAITRS IADQARTIRI PVHMIETINK LNRVSRQMIQB1-32597MKAVDKFEYR RGYKFSTYAT WWIRQAITRS IADQARTIRI PVHMIETINK LNRVSRQMIQ EM-90MKAVDKFEYR RGYKFSTYAT WWIRQAITRS IADQARTIRI PVHMIETINK LNRVSRQMIQ LF-89MKAVDKFEYR RGYKFSTYAT WWIRQAITRS IADQARTIRI PVHMIETINK LNRVSRQMIQAL10016---------- ---------- ---------- ---------- -VHMIETINK LNRVSRQMIQAL10008---------- ---------- ---------- ---------- -VHMIETINK LNRVSRQMIQAL20218---------- ---------- ---------- ---------- -VHMIETINK LNRVSRQMIQAL20219---------- ---------- ---------- ---------- -VHMIETINK LNRVSRQMIQAL20222---------- ---------- ---------- ---------- -VHMIETINK LNRVSRQMIQAL20223---------- ---------- ---------- ---------- -VHMIETINK LNRVSRQMIQAL20277---------- ---------- ---------- ---------- -VHMIETINK LNRVSRQMIQ 481PHARMAQ_001ELGREATPEE LSERMEMPEH KIRKILKIAK EPISMETPIG DDEDSHLGDF IEDTTMQLPVA1-15972ELGREATPEE LSERMEMPEH KIRKILKIAK EPISMETPIG DDEDSHLGDF IEDTTMQLPVB1-32597ELGREATPEE LSERMEMPEH KIRKILKIAK EPISMETPIG DDEDSHLGDF IEDTTMQLPV EM-90ELGREATPEE LSERMEMPEH KIRKILKIAK EPISMETPIG DDEDSHLGDF IEDTTMQLPV LF-89ELGREATPEE LSERMEMPEH KIRKILKIAK EPISMETPIG DDEDSHLGDF IEDTTMQLPVAL10016ELGREATPEE LSERMEMPEH KIRK------ ---------- ---------- ----------AL10008ELGREATPEE LSERMEMPEH KIRK------ ---------- ---------- ----------AL20218ELGREATPEE LSERMEMPEH KIRK------ ---------- ---------- ----------AL20219ELGREATPEE LSERMEMPEH KIRK------ ---------- ---------- ----------AL20222ELGREATPEE LSERMEMPEH KIRK------ ---------- ---------- ----------AL20223ELGREATPEE LSERMEMPEH KIRK------ ---------- ---------- ----------AL20277ELGREATPEE LSERMEMPEH KIRK------ ---------- ---------- ---------- 541PHARMAQ_001DSTMGDALKQ ATSDILENLT PREAEVLRMR FGIDMNTDHT LEEVGKQFDV TRERIRQIEAA1-15972DSTMGDALKQ ATSDILENLT PREAKVLRMR FGIDMNTDHT LEEVGKQFDV TRERIRQIEAB1-32597DSTMGDALKQ ATSDILENLT PREAKVLRMR FGIDMNTDHT LEEVGKQFDV TRERIRQIEA EM-90DSTMGDALKQ ATSDILENLT PREAKVLRMR FGIDMNTDHT LEEVGKQFDV TRERIRQIEA LF-89DSTMGDALKQ ATSDILENLT PREAKVLRMR FGIDMNTDHT LEEVGKQFDV TRERIRQIEAAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20222---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20277---------- ---------- ---------- ---------- ---------- ---------- 601PHARMAQ_001 KALRKLRHPT RSEILKSFLD SEE* (Seq. ID No. 13) A1-15972KALRKLRHPT RSEILKSFLD SEE* (Seq. ID No. 14) B1-32597KALRKLRHPT RSEILKSFLD SEE* (Seq. ID No. 15) EM-90KALRKLRHPT RSEILKSFLD SEE* (Seq. ID No. 16) LF-89KALRKLRHPT RSEILKSFLD SEE* (Seq. ID No. 17) AL10016---------- ---------- ---- (Seq. ID No. 18) AL10008---------- ---------- ---- (Seq. ID No. 19) AL20218---------- ---------- ---- (Seq. ID No. 20) AL20219---------- ---------- ---- (Seq. ID No. 21) AL20222---------- ---------- ---- (Seq. ID No. 22) AL20223---------- ---------- ---- (Seq. ID No. 23) AL20277---------- ---------- ---- (Seq. ID No. 24)

The rpoD amino acid sequence alignment reveals that there are naturalpolymorphisms of the rpoD gene product between wild-type strains.However, all of the wild-type strains are virulent and therefore none ofthe differences between the wild-type sequences can be considered toaffect virulence. PHARMAQ 001 has an arginine to cysteine mutation atposition 473 of the amino acid sequence of the rpoD gene product andthis mutation is not seen in any of the wild-type strains investigated,such as A1-15972, B1-32597, EM-90, and/or LF-89. The protein sequence ofthe rpoD gene product in PHARMAQ 001 is otherwise identical to that ofwild-type strains including A1-15972, EM-90, and LF-89. The sequences of11 wild-type strains were examined in the region of amino acid residues462-504 of the rpoD gene product. All of the sequences were found to beidentical in this region, but different to that of PHARMAQ 001.

FecR amino acid sequence alignment:

PHARMAQ_001MKINHQPGGI MLIMNNHGEV MTSYSHMMIF FSNYGEKVKI ENQRILNDNK LLFSNRVSRV LF-89MKINHQPGGI MLIMNNHGEV MTSYSHMMSF FSNYGEKVKI ENQRILNDNK LLFSNRVSRV EM-90MKINHQPGGI MLIMNNHGEV MTSYSHMMSF FSNYGEKVKI ENQRILNDNK LLFSNRVSRVB1-32597MKINHQPGGI MLIMNNHGEV MTSYSHMMSF FSNYGEKVKI ENQRILNDNK LLFSNRVSEVA1-15972MKINHQPGGI MLIMNNHGEV MTSYSHMMIF FSNYGEKVKI ENQRILNDNK LLFSNRVSRVAL10016---------- ---------- ---------- -------?KI ENQRILNDNK LLFSNRVSRVAL10008---------- ---------- ---------- -------?KI ENQRILNDNK LLFSNRVSRVAL20218---------- ---------- ---------- -------?KI ENQRILNDNK LLFSNRVSRVAL20219---------- ---------- ---------- -------?KI ENQRILNDNK LLFSNRVSRVAL20220---------- ---------- ---------- -------?KI ENQRILNDNK LLFSNRVSEVAL20223---------- ---------- ---------- -------?KI ENQRILNDNK LLFSNRVSEVAL20470---------- ---------- ---------- -------?KI ENQRILNDNK LLFSNRVSEVAL20471---------- ---------- ---------- -------?KI ENQRILNDNK LLFSNRVSEVAL20455---------- ---------- ---------- -------?KI ENQRILNDNK LLFSNRVSEV 61PHARMAQ_001 RYRPCLIIDA KDALSVCSGV FH

------- ---------- ---------- ---------- LF-89RYRPCLIIDA KDALSVCSGV FHQVKNEFGV VVASSLNVMI YDYKSMSDED IIHILKSVKK EM-90RYRPCLIIDA KDALSVCSGV FHQVKNEFGV VVASSLNVMI YDYKSMSDED IIHILKSVKKB1-32597RYRPCLIIDA KDAFSVCSGV FHQVKNEFGV VVANSLNVMI YDYKSMSDED IIHILKSVKKA1-15972RYRPCLIIDA KDALSVCSGV FHQVKNEFGV VVASSLNVMI YDYKSMSDED IIHILKSVKKAL10016RYRPCLIIDA KDALSVCSGV FHQVKNEFGV VVASSLNVMI YDYKSMSDED IIHILKSVKKAL10008RYRPCLIIDA KDALSVCSGV FHQVKNEFGV VVASSLNVMI YDYKSMSDED IIHILKSVKKAL20218RYRPCLIIDA KDALSVCSGV FHQVKNEFGV VVASSLNVMI YDYKSMSDED IIHILKSVKKAL20219RYRPCLIIDA KDALSVCSGV FHQVKNEFGV VVASSLNVMI YDYKSMSDED IIHILKSVKKAL20220RYRPCLIIDA KDAFSVCSGV FHQVKNEFGV VVANSLNVMI YDYKSMSDED IIHILKSVKKAL20223RYRPCLIIDA KDAFSVCSGV FHQVKNEFGV VVANSLNVMI YDYKSMSDED IIHILKSVKKAL20470RYRPCLIIDA KDAFSVCSGV FHQVKNEFGV VVANSLNVMI YDYKSMSDED IIHILKSVKKAL20471RYRPCLIIDA KDAFSVCSGV FHQVKNEFGV VVANSLNVMI YDYKSMSDED IIHILKSVKKAL20455RYRPCLIIDA KDAFSVCSGV FHQVKNEFGV VVANSLNVMI YDYKSMSDED IIHILKSVKK 121PHARMAQ_001---------- ---------- ---------- ---------- ---------- ---------- LF-89HPKLSLIESK ILFLKVVMKG IKCRHIESLL KVSGSTVYTY CMNIKSKANI FSFKGQSVIQ EM-90HPKLSLIESK ILFLKVVMKG IKCRHIESLL KVSGSTVYTY CMNIKSKANI FSFKGQSVIQB1-32597HPKLSLIESK ILFLKVVMKG IKCRHIESLL KVSGSTVYTY CMNIKSKANI FSFKGQSVIQA1-15972HPKLSLIESK ILFLKVVMKG IKCRHIESLL KVSGSTVYTY CMNIKSKANI FSFKGQSVIQAL10016HPKLSLIESK ILFLKVV?-- ---------- ---------- ---------- ----------AL10008HPKLSLIESK ILFLKVV?-- ---------- ---------- ---------- ----------AL20218HPKLSLIESK ILFLKVV?-- ---------- ---------- ---------- ----------AL20219HPKLSLIESK ILFLKVV?-- ---------- ---------- ---------- ----------AL20220HPKLSLIESK ILFLKVV?-- ---------- ---------- ---------- ----------AL20223HPKLSLIESK ILFLKVV?-- ---------- ---------- ---------- ----------AL20470HPKLSLIESK ILFLKVV?-- ---------- ---------- ---------- ----------AL20471HPKLSLIESK ILFLKVV?-- ---------- ---------- ---------- ----------AL20455HPKLSLIESK ILFLKVV?-- ---------- ---------- ---------- ---------- 181PHARMAQ_001 ---------- ---------- ---------- --------- (Seq. ID No. 25)LF-89 ELEKSQFFSD ISIKINIDFY NINNGVNEKN VCQVYSLA* (Seq. ID No. 26) EM-90ELEKSQFFSD ISIKINIDFY NINNGVNEKN VCQVYSLA* (Seq. ID No. 27) B1-32597ELEKSQFFSD IAMKINIDFY SINNEANEKN VCQVYSLA* (Seq. ID No. 28) A1-15972ELEKSQFFSD ISIKINIDFY NINNGVNEKN VCQVYSLA* (Seq. ID No. 29) AL10016---------- ---------- ---------- --------- (Seq. ID No. 30) AL10008---------- ---------- ---------- --------- (Seq. ID No. 31) AL20218---------- ---------- ---------- --------- (Seq. ID No. 32) AL20219---------- ---------- ---------- --------- (Seq. ID No. 33) AL20220---------- ---------- ---------- --------- (Seq. ID No. 34) AL20223---------- ---------- ---------- --------- (Seq. ID No. 35) AL20470---------- ---------- ---------- --------- (Seq. ID No. 36) AL20471---------- ---------- ---------- --------- (Seq. ID No. 37) AL20455---------- ---------- ---------- --------- (Seq. ID No. 38)

The FecR amino acid sequence alignment reveals that there are naturalpolymorphisms of the FecR gene product between wild-type strains.However, all of the wild-type strains are virulent and therefore none ofthe differences between the wild-type sequences can be considered toaffect virulence. PHARMAQ 001 has a premature stop codon introduced atposition 83 of the amino acid, and this mutation is not seen in any ofthe wild-type strains investigated, such as A1-15972, B1-32597, EM-90,and/or LF-89. The protein sequence of the FecR gene product in PHARMAQ001 is otherwise identical to that of wild-type strains includingA1-15972, EM-90, and LF-89. The sequences of 13 wild-type strains wereexamined in the region of amino acid residues 39-137 of the FecR geneproduct. None of the wild-type sequences were found to be prematurelytruncated, whereas in contrast, the FecR gene product of PHARMAQ 001 isterminated by a stop codon in position 83.

ATP-grasp domain protein amino acid sequence alignment:

PHARMAQ001MISLWKTYQA LKTKGILGIN QRNADFIIRY NQRKYYPLVD DKIMTKTLAI KDGIAVPKLY LF-89MISLWKTYQA LKTKGILGIN QRNADFIIRY NQRKYYPLVD DKIMTKTLAI KDGIAVPKLY EM-90MISLWKTYQA LKTKGILGIN QRNADFIIRY NQRKYYPLVD DKIMTKTLAI KDGIAVPKLYB1-32597MISLWKTYQA LKTKGILGIN QRNADFIIRY NQRKYYPLVD DKIMTKTLAI KDGIAVPKLYA1-15972MISLWKTYQA LKTKGILGIN QRNADFIIRY NQRKYYPLVD DKIMTKTLAI KDGIAVPKLYAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20220---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20470---------- ---------- ---------- ---------- ---------- ----------AL20471---------- ---------- ---------- ---------- ---------- ----------AL20455---------- ---------- ---------- ---------- ---------- ---------- 61PHARMAQ001ATLKTDHDTH HLEQILANRT DFVIKPARGA GGDGILVITN RHGERFRKVS GALLHLDDIR LF-89ATLKTDHDTH HLEQILANRT DFVIKPARGA GGDGILVITN RHGERFRKVS GALLHLDDIR EM-90ATLKTDHDTH HLEQILANRT DFVIKPARGA GGDGILVITN RHGERFRKVS GALLHLDDIRB1-32597ATLKTDHDTH HLEQILANRT DFVIKPARGA GGDGILVITN RHGERFRKVS GALLHLDDIRA1-15972ATLKTDHDTH HLEQILANRT DFVIKPARGA GGDGILVITN RHGERFRKVS GALLHLDDIRAL10016---------- ---------- ---------- ---------- ---------- ------?DIRAL10008---------- ---------- ---------- ---------- ---------- ------?DIRAL20218---------- ---------- ---------- ---------- ---------- ------?DIRAL20219---------- ---------- ---------- ---------- ---------- ------?DIRAL20220---------- ---------- ---------- ---------- ---------- ------?DIRAL20223---------- ---------- ---------- ---------- ---------- ------?DIRAL20470---------- ---------- ---------- ---------- ---------- ------?DIRAL20471---------- ---------- ---------- ---------- ---------- ------?DIRAL20455---------- ---------- ---------- ---------- ---------- ------?DIR 121PHARMAQ001HHISNILSGV YSLGGQRDQA MIEYRVQFDP LFKKISYQGV PDIRIIVLKG YPAMAMVRLP LF-89HHISNILSGV YSLGGQRDQA MIEYRVQFDP LFKKISYQGV PDIRIIVLKG YPAMAMVRLP EM-90HHISNILSGV YSLGGQRDQA MIEYRVQFDP LFKKISYQGV PDIRIIVLKG YPAMAMVRLPB1-32597HHISNILSGV YSLGGQRDQA MIEYRVQFDP LFKKISYQGV PDIRIIVLKG YPAMAMVRLPA1-15972HHISNILSGV YSLGGQRDQA MIEYRVQFDP LFKKISYQGV PDIRIIVLKG YPAMAMVRLPAL10016HHISNILSGV YSLGGQRDQA MIEYRVQFDP LFKKISYQGV PDIRIIVLKG YPAMAMVRLPAL10008HHISNILSGV YSLGGQRDQA MIEYRVQFDP LFKKISYQGV PDIRIIVLKG YPAMAMVRLPAL20218HHISNILSGV YSLGGQRDQA MIEYRVQFDP LFKKISYQGV PDIRIIVLKG YPAMAMVRLPAL20219HHISNILSGV YSLGGQRDQA MIEYRVQFDP LFKKISYQGV PDIRIIVLKG YPAMAMVRLPAL20220HHISNILSGV YSLGGQRDQA MIEYRVQFDP LFKKISYQGV PDIRIIVLKG YPAMAMVRLPAL20223HHISNILSGV YSLGGQRDQA MIEYRVQFDP LFKKISYQGV PDIRIIVLKG YPAMAMVRLPAL20470HHISNILSGV YSLGGQRDQA MIEYRVQFDP LFKKISYQGV PDIRIIVLKG YPAMAMVRLPAL20471HHISNILSGV YSLGGQRDQA MIEYRVQFDP LFKKISYQGV PDIRIIVLKG YPAMAMVRLPAL20455HHISNILSGV YSLGGQRDQA MIEYRVQFDP LFKKISYQGV PDIRIIVLKG YPAMAMVRLP 181   * PHARMAQ001 TRL PDGKANL HQGAIGVGID LTTGITLEGV WMNDPIHEHP DTGYAVPGLQ IPHWDHFLNL LF-89TRLSDGKANL HQGAIGVGID LTTGITLEGV WMNDPIHEHP DTGYAVPGLQ IPHWDHFLNL EM-90TRLSDGKANL HQGAIGVGID LTTGITLEGV WMNDPIHEHP DTGYAVPGLQ IPHWDHFLNLB1-32597TRLSDGKANL HQGAIGVGID LTTGITLEGV WMNDPIHEHP DTGYAVPGLQ IPHWDHFLNLA1-15972TRLSDGKANL HQGAIGVGID LTTGITLEGV WMNDPIHEHP DTGYAVPGLQ IPHWDHFLNLAL10016TRLSDGKANL HQGAIGVGID LTTGITLEGV WMNDPIHEHP DTGYAVPGLQ IPHWDHFLNLAL10008TRLSDGKANL HQGAIGVGID LTTGITLEGV WMNDPIHEHP DTGYAVPGLQ IPHWDHFLNLAL20218TRLSDGKANL HQGAIGVGID LTTGITLEGV WMNDPIHEHP DTGYAVPGLQ IPHWDHFLNLAL20219TRLSDGKANL HQGAIGVGID LTTGITLEGV WMNDPIHEHP DTGYAVPGLQ IPHWDHFLNLAL20220TRLSDGKANL HQGAIGVGID LTTGITLEGV WMNDPIHEHP DTGYAVPGLQ IPHWDHFLNLAL20223TRLSDGKANL HQGAIGVGID LTTGITLEGV WMNDPIHEHP DTGYAVPGLQ IPHWDHFLNLAL20470TRLSDGKANL HQGAIGVGID LTTGITLEGV WMNDPIHEHP DTGYAVPGLQ IPHWDHFLNLAL20471TRLSDGKANL HQGAIGVGID LTTGITLEGV WMNDPIHEHP DTGYAVPGLQ IPHWDHFLNLAL20455TRLSDGKANL HQGAIGVGID LTTGITLEGV WMNDPIHEHP DTGYAVPGLQ IPHWDHFLNL 241PHARMAQ001AARCYELTQL GYLGVDIILD KDKGPLMLEL NARPGLNIQI ANNSGLLHRL RFIEQQNQQR LF-89AARCYELTQL GYLGVDIILD KDKGPLMLEL NARPGLNIQI ANNSGLLHRL RFIEQQNQQR EM-90AARCYELTQL GYLGVDIILD KDKGPLMLEL NARPGLNIQI ANNSGLLHRL RFIEQQNQQRB1-32597AARCYELTQL GYLGVDIILD KDKGPLMLEL NARPGLNIQI ANNSGLLHRL RFIEQQNQQRA1-15972AARCYELTQL GYLGVDIILD KDKGPLMLEL NARPGLNIQI ANNSGLLHRL RFIEQQNQQRAL10016AARCYELTQL G?-------- ---------- ---------- ---------- ----------AL10008AARCYELTQL G?-------- ---------- ---------- ---------- ----------AL20218AARCYELTQL G?-------- ---------- ---------- ---------- ----------AL20219AARCYELTQL G?-------- ---------- ---------- ---------- ----------AL20220AARCYELTQL G?-------- ---------- ---------- ---------- ----------AL20223AARCYELTQL G?-------- ---------- ---------- ---------- ----------AL20470AARCYELTQL G?-------- ---------- ---------- ---------- ----------AL20471AARCYELTQL G?-------- ---------- ---------- ---------- ----------AL20455AARCYELTQL G?-------- ---------- ---------- ---------- ---------- 301PHARMAQ001 TADERIAFIK HQFAKI* (Seq. ID No. 39) LF-89TADERIAFIK HQFAKI* (Seq. ID No. 40) EM-90TADERIAFIK HQFAKI* (Seq. ID No. 41) B1-32597TADERIAFIK HQFAKI* (Seq. ID No. 42) A1-15972TADERIAFIK HQFAKI* (Seq. ID No. 43) AL10016---------- ------- (Seq. ID No. 44) AL10008---------- ------- (Seq. ID No. 45) AL20218---------- ------- (Seq. ID No. 46) AL20219---------- ------- (Seq. ID No. 47) AL20220---------- ------- (Seq. ID No. 48) AL20223---------- ------- (Seq. ID No. 49) AL20470---------- ------- (Seq. ID No. 50) AL20471---------- ------- (Seq. ID No. 51) AL20455---------- ------- (Seq. ID No. 52)

All of the wild-type sequences of the ATP-grasp domain protein geneproduct that were 50 investigated were found to be identical. However,PHARMAQ 001 has a serine to proline mutation at position 184 of theamino acid sequence of the ATP-grasp domain protein gene product whichis not seen in the wild-type sequence. The sequences of 13 wild-typestrains were examined in the region of amino acid residues 118-251 ofthe ATP-grasp domain protein gene product. All of the sequences werefound to be 55 identical in this region, but different to that ofPHARMAQ 001.

FtsH amino acid sequence alignment:

PHARMAQ001MIKNIMLWLV IALVLVTVFS NLGPRQQSVN RLDYSTFVKD INNGQVKSVI IDGLNIKGQT LF-89MIKNIMLWLV IALVLVTVFS NLGPRQQSVN RLDYSTFVKD INNGQVKSVI IDGLNIKGQT EM-90MIKNIMLWLV IALVLVTVFS NLGPRQQSVN RLDYSTFVKD INNGQVKSVI IDGLNIKGQTB1_32597MIKNIMLWLV IALVLVTVFS NLGPRQQSVN RLDYSTFVKD INNGQVKSVI IDGLNIKGQTA1_15972MIKNIMLWLV IALVLVTVFS NLGPRQQSVN RLDYSTFVKD INNGQVKSVI IDGLNIKGQTAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20220---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20470---------- ---------- ---------- ---------- ---------- ----------AL20471---------- ---------- ---------- ---------- ---------- ----------AL20455---------- ---------- ---------- ---------- ---------- ---------- 61PHARMAQ001SSGTPFATYI PWKDPFLMDQ MLAKNVTIAA KPPEQRSWLL SALISWFPGI LLIAIWIFFL LF-89SSGTPFATYI PWKDPFLMDQ MLAKNVTIAA KPPEQRSWLL SALISWFPGI LLIAIWIFFL EM-90SSGTPFATYI PWKDPFLMDQ MLAKNVTIAA KPPEQRSWLL SALISWFPGI LLIAIWIFFLB1_32597SSGTPFATYI PWKDPFLMDQ MLSKNVTIAA KPPEQRSWLL SALISWFPGI LLIAIWIFFLA1_15972SSGTPFATYI PWKDPFLMDQ MLAKNVTIAA KPPEQRSWLL SALISWFPGI LLIAIWIFFLAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20220---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20470---------- ---------- ---------- ---------- ---------- ----------AL20471---------- ---------- ---------- ---------- ---------- ----------AL20455---------- ---------- ---------- ---------- ---------- ---------- 121PHARMAQ001RQMQGGGGGK GMMSFGSSKA RLLGEDQIKV NFADVAGCEE AKEEVKELVD FLRDPTKFQK LF-89RQMQGGGGGK GMMSFGSSKA RLLGEDQIKV NFADVAGCEE AKEEVKELVD FLRDPTKFQK EM-90RQMQGGGGGK GMMSFGSSKA RLLGEDQIKV NFADVAGCEE AKEEVKELVD FLRDPTKFQKB1_32597RQMQGGGGGK GMMSFGSSKA RLLGEDQIKV NFADVAGCEE AKEEVKELVD FLRDPTKFQKA1_15972RQMQGGGGGK GMMSFGSSKA RLLGEDQIKV NFADVAGCEE AKEEVKELVD FLRDPTKFQKAL10016---------- ---------- ---------- -FADVAGCEE AKEEVKELVD FLRDPTKFQKAL10008---------- ---------- ---------- -FADVAGCEE AKEEVKELVD FLRDPTKFQKAL20218---------- ---------- ---------- -FADVAGCEE AKEEVKELVD FLRDPTKFQKAL20219---------- ---------- ---------- -FADVAGCEE AKEEVKELVD FLRDPTKFQKAL20220---------- ---------- ---------- -FADVAGCEE AKEEVKELVD FLRDPTKFQKAL20223---------- ---------- ---------- -FADVAGCEE AKEEVKELVD FLRDPTKFQKAL20470---------- ---------- ---------- -FADVAGCEE AKEEVKELVD FLRDPTKFQKAL20471---------- ---------- ---------- -FADVAGCEE AKEEVKELVD FLRDPTKFQKAL20455---------- ---------- ---------- -FADVAGCEE AKEEVKELVD FLRDPTKFQK 181PHARMAQ001 LGGKIPQGVL 

VGPPGTGKT LLAKAIAGEA KVPFFSISGS DFVEMFVGVG ASRVRDMFDQ LF-89LGGKIPQGVL MVGPPGTGKT LLAKAIAGEA KVPFFSISGS DFVEMFVGVG ASRVRDMFDQ EM-90LGGKIPQGVL MVGPPGTGKT LLAKAIAGEA KVPFFSISGS DFVEMFVGVG ASRVRDMFDQB1_32597LGGKIPQGVL MVGPPGTGKT LLAKAIAGEA KVPFFSISGS DFVEMFVGVG ASRVRDMFDQA1_15972LGGKIPQGVL MVGPPGTGKT LLAKAIAGEA KVPFFSISGS DFVEMFVGVG ASRVRDMFDQAL10016LGGKIPQGVL MVGPPGTGKT LLAKAIAGEA KVPFFSISGS DFVEMFVGVG ASRVRDMFDQAL10008LGGKIPQGVL MVGPPGTGKT LLAKAIAGEA KVPFFSISGS DFVEMFVGVG ASRVRDMFDQAL20218LGGKIPQGVL MVGPPGTGKT LLAKAIAGEA KVPFFSISGS DFVEMFVGVG ASRVRDMFDQAL20219LGGKIPQGVL MVGPPGTGKT LLAKAIAGEA KVPFFSISGS DFVEMFVGVG ASRVRDMFDQAL20220LGGKIPQGVL MVGPPGTGKT LLAKAIAGEA KVPFFSISGS DFVEMFVGVG ASRVRDMFDQAL20223LGGKIPQGVL MVGPPGTGKT LLAKAIAGEA KVPFFSISGS DFVEMFVGVG ASRVRDMFDQAL20470LGGKIPQGVL MVGPPGTGKT LLAKAIAGEA KVPFFSISGS DFVEMFVGVG ASRVRDMFDQAL20471LGGKIPQGVL MVGPPGTGKT LLAKAIAGEA KVPFFSISGS DFVEMFVGVG ASRVRDMFDQAL20455LGGKIPQGVL MVGPPGTGKT LLAKAIAGEA KVPFFSISGS DFVEMFVGVG ASRVRDMFDQ 241PHARMAQ001AKKRAPCIIF IDEIDAVGRH RGSGMGGGHD EREQTLNQML VEMDGFEGTE GVIVIAATNR LF-89AKKRAPCIIF IDEIDAVGRH RGSGMGGGHD EREQTLNQML VEMDGFEGTE GVIVIAATNR EM-90AKKRAPCIIF IDEIDAVGRH RGSGMGGGHD EREQTLNQML VEMDGFEGTE GVIVIAATNRB1_32597AKKRAPCIIF IDEIDAVGRH RGSGMGGGHD EREQTLNQML VEMDGFEGTE GVIVIAATNRA1_15972AKKRAPCIIF IDEIDAVGRH RGSGMGGGHD EREQTLNQML VEMDGFEGTE GVIVIAATNRAL10016AKKRAPCIIF IDEIDAVGRH RGSGMGGGHD EREQ?----- ---------- ----------AL10008AKKRAPCIIF IDEIDAVGRH RGSGMGGGHD EREQ?----- ---------- ----------AL20218AKKRAPCIIF IDEIDAVGRH RGSGMGGGHD EREQ?----- ---------- ----------AL20219AKKRAPCIIF IDEIDAVGRH RGSGMGGGHD EREQ?----- ---------- ----------AL20220AKKRAPCIIF IDEIDAVGRH RGSGMGGGHD EREQ?----- ---------- ----------AL20223AKKRAPCIIF IDEIDAVGRH RGSGMGGGHD EREQ?----- ---------- ----------AL20470AKKRAPCIIF IDEIDAVGRH RGSGMGGGHD EREQ?----- ---------- ----------AL20471AKKRAPCIIF IDEIDAVGRH RGSGMGGGHD EREQ?----- ---------- ----------AL20455AKKRAPCIIF IDEIDAVGRH RGSGMGGGHD EREQ?----- ---------- ---------- 301PHARMAQ001PDVLDPALLR PGRFDRQVSV GLPDVKGREQ ILKVHMRKVP LGDDVKASLI ARGTPGFSGA LF-89PDVLDPALLR PGRFDRQVSV GLPDVKGREQ ILKVHMRKVP LGDDVKASLI ARGTPGFSGA EM-90PDVLDPALLR PGRFDRQVSV GLPDVKGREQ ILKVHMRKVP LGDDVKASLI ARGTPGFSGAB1_32597PDVLDPALLR PGRFDRQVSV GLPDVKGREQ ILKVHMRKVP LGDDVKASLI ARGTPGFSGAA1_15972PDVLDPALLR PGRFDRQVSV GLPDVKGREQ ILKVHMRKVP LGDDVKASLI ARGTPGFSGAAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20220---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20470---------- ---------- ---------- ---------- ---------- ----------AL20471---------- ---------- ---------- ---------- ---------- ----------AL20455---------- ---------- ---------- ---------- ---------- ---------- 361PHARMAQ001DLANLVNEAA LFAARKDKTV VAMREFDDAK DKILMGTERR SMAMTEEQKR LTAFHEAGHA LF-89DLANLVNEAA LFAARKDKTV VAMREFDDAK DKILMGTERR SMAMTEEQKR LTAFHEAGHA EM-90DLANLVNEAA LFAARKDKTV VAMREFDDAK DKILMGTERR SMAMTEEQKR LTAFHEAGHAB1_32597DLANLVNEAA LFAARKDKTV VAMREFDDAK DKILMGTERR SMAMTEEQKR LTAFHEAGHAA1_15972DLANLVNEAA LFAARKDKTV VAMREFDDAK DKILMGTERR SMAMTEEQKR LTAFHEAGHAAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20220---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20470---------- ---------- ---------- ---------- ---------- ----------AL20471---------- ---------- ---------- ---------- ---------- ----------AL20455---------- ---------- ---------- ---------- ---------- ---------- 421PHARMAQ001IVGCLVPDHD PVYKVSIVPR GRALGVTMYL PEEDSYGYSR ERLESLISSM YGGRIAEALV LF-89IVGCLVPDHD PVYKVSIVPR GRALGVTMYL PEEDSYGYSR ERLESLISSM YGGRIAEALV EM-90IVGCLVPDHD PVYKVSIVPR GRALGVTMYL PEEDSYGYSR ERLESLISSM YGGRIAEALVB1_32597IVGCLVPDHD PVYKVSIVPR GRALGVTMYL PEEDSYGYSR ERLESLISSM YGGRIAEALVA1_15972IVGCLVPDHD PVYKVSIVPR GRALGVTMYL PEEDSYGYSR ERLESLISSM YGGRIAEALVAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20220---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20470---------- ---------- ---------- ---------- ---------- ----------AL20471---------- ---------- ---------- ---------- ---------- ----------AL20455---------- ---------- ---------- ---------- ---------- ---------- 481PHARMAQ001FGVEKVTTGA SNDIEKASEV ARNMVTKWGL SERLGPILYG QEGGDPFGYG AGKGTPEFSD LF-89FGVEKVTTGA SNDIEKASEV ARNMVTKWGL SERLGPILYG QEGGDPFGYG AGKGTPEFSD EM-90FGVEKVTTGA SNDIEKASEV ARNMVTKWGL SERLGPILYG QEGGDPFGYG AGKGTPEFSDB1_32597FGVEKVTTGA SNDIEKASEV ARNMVTKWGL SERLGPILYG QEGGDPFGYG AGKGTPEFSDA1_15972FGVEKVTTGA SNDIEKASEV ARNMVTKWGL SERLGPILYG QEGGDPFGYG AGKGTPEFSDAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20220---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20470---------- ---------- ---------- ---------- ---------- ----------AL20471---------- ---------- ---------- ---------- ---------- ----------AL20455---------- ---------- ---------- ---------- ---------- ---------- 541PHARMAQ001QTSVAIDEEV RQIIDRNYTR AESILINNRD ILDAMADALM VYETIDRDQV ADLMARRPVK LF-89QTSVAIDEEV RQIIDRNYTR AESILINNRD ILDAMADALM VYETIDRDQV ADLMARRPVK EM-90QTSVAIDEEV RQIIDRNYTR AESILINNRD ILDAMADALM VYETIDRDQV ADLMARRPVKB1_32597QTSVAIDEEV RQIIDRNYTR AESILIDNRD ILDAMADALM VYETIDREQV ADLMARRPVKA1_15972QTSVAIDEEV RQIIDRNYTR AESILINNRD ILDAMADALM VYETIDRDQV ADLMARRPVKAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20220---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20470---------- ---------- ---------- ---------- ---------- ----------AL20471---------- ---------- ---------- ---------- ---------- ----------AL20455---------- ---------- ---------- ---------- ---------- ---------- 601PHARMAQ001APKDWDQPSD ESGSSASGDE LQPLDANINT DINETKSADQ ETDQGAPSPE IKGKPADDPT LF-89APKDWDQPSD ESGSSASGDE LQPLDANINT DINETKSADQ ETDQGAPSPE IKGKPADDPT EM-90APKDWDQPSD ESGSSASGDE LQPLDANINT DINETKSADQ ETDQGAPSPE IKGKPADDPTB1_32597APKDWDQPSD ESGSSASGDE LQPLDANINT DINDTKSADQ EIDQGAPSPE IKGKPADDPTA1_15972APKDWDQPSD ESGSSASGDE LQPLDANINT DINETKSADQ ETDQGAPSPE IKGKPADDPTAL10016---------- ---------- ---------- ---------- ---------- ----------AL10008---------- ---------- ---------- ---------- ---------- ----------AL20218---------- ---------- ---------- ---------- ---------- ----------AL20219---------- ---------- ---------- ---------- ---------- ----------AL20220---------- ---------- ---------- ---------- ---------- ----------AL20223---------- ---------- ---------- ---------- ---------- ----------AL20470---------- ---------- ---------- ---------- ---------- ----------AL20471---------- ---------- ---------- ---------- ---------- ----------AL20455---------- ---------- ---------- ---------- ---------- ---------- 661PHARMAQ001 * (Seq. ID No. 53) LF-89 * (Seq. ID No. 54) EM-90* (Seq. ID No. 55) B1_32597 * (Seq. ID No. 56) A1_15972* (Seq. ID No. 57) AL10016 - (Seq. ID No. 58) AL10008 - (Seq. ID No. 59)AL20218 - (Seq. ID No. 60) AL20219 - (Seq. ID No. 61) AL20220- (Seq. ID No. 62) AL20223 - (Seq. ID No. 63) AL20470 - (Seq. ID No. 64)AL20471 - (Seq. ID No. 65) AL20455 - (Seq. ID No. 66)

The FtsH amino acid sequence alignment reveals that there are naturalpolymorphisms of the FtsH gene product between wild-type strains.However, all of the wild-type strains are virulent and therefore none ofthe differences between the wild-type sequences can be considered toaffect virulence. PHARMAQ 001 has a methionine to isoleucine mutation atposition 191 of the amino acid sequence which is not seen in any of thewild-type strains investigated, such as A1-15972, B1-32597, EM-90,and/or LF-89. The protein sequence of the FtsH gene product in PHARMAQ001 is otherwise identical to that of wild-type strains includingA1-15972, EM-90, and LF-89. The sequences of 13 wild-type strains wereexamined in the region of amino acid residues 152-274 of the FtsH geneproduct. All of the sequences were found to be identical in this region,but different to that of PHARMAQ 001.

It is clear from the alignments that P. salmonis gene products are veryhighly conserved in all of the strains investigated. As would beexpected, there are natural polymorphisms in the genes wherein some ofthe wild-type strains have a sequence that is different from that ofother wild-type strains. However, since all the strains except PHARMAQ001 are virulent, these differences cannot contribute to the loss ofvirulence and consequent attenuated phenotype observed in PHARMAQ 001.

PHARMAQ 001 has mutations in the amino acid sequence of the rpoD, FecR,ATP-grasp domain protein, and FtsH gene products relative to thewild-type sequence, and also has an attenuated phenotype. Thesemutations are the only mutations observed in PHARMAQ 001 which lead to asignificant alteration in the amino acid sequence of a protein, and theyare not observed in any of the virulent strains investigated.

In a P. salmonis strain with an attenuated phenotype, if a mutation isobserved in one of the rpoD, FecR, ATP-grasp domain protein, or FtsHgenes which is also present in a virulent wild-type strain, then themutation cannot be responsible for the attenuated phenotype. Mutations,and in particular attenuating mutations, in the rpoD, FecR, ATP-graspdomain protein, or FtsH genes are therefore only significant if theylead to a difference in the amino acid sequence of the gene productrelative to the protein sequence of one of the virulent wild-typestrains, such as A1-15972, B1-32597, EM-90, and/or LF-89.

Example 3: Phylogeny of Piscirickettsia salmonis

To classify the Piscirickettsia salmonis isolates in Table 1, asimplified MLSA-scheme was used employing the genetic information withinthe arginine N-succinyltransferase (ast), glutamate-1-semialdehydeaminotransferase (hemL), L-serine dehydratase (sdhL), andUDP-glucose-4-epimerase (galE) genes for phylogenetic predictions (asannotated in the genome of A1-15972). Sequence information was obtainedby standard PCR and sequencing. DNA sequences were assembled, qualitychecked and trimmed using Vector NTI® software. Sequences for thestrains A1-15972, B1-32597, LF-89 and EM-90 were retrieved from GenBank.Sequence information from the four genes for each strain were trimmedand concatenated before alignment.

Sequence alignments and phylogenetic predictions were performed inMEGA5, as described in Tamura et al. (Mol Biol Evol. 2011 October;28(10): 2731-9, incorporated herein by reference), with the followingparameters:

Statistical Method: Maximum Likelihood (100 Bootstrap Replications)

Substitution Model: Tamura-Nei model

Rates: Gamma distributed with Invariant sites (16 Discrete GammaCategories)

Upon phylogenetic analysis of DNA sequences from the four genes above,two distinct and separate genotypes were identified. The two genotypesare here referred to as the LF-89 group and the EM-90 group. PHARMAQ001l was found to group together with the EM-90 group. The LF-89sequence used in the preparation of the phylogenetic tree was thegenomic sequence deposited under GenBank accession no. AMGC00000000.1.

Example 4: Avirulence of Isolate PHARMAQ 001l when Used as a LiveAttenuated Vaccine

PHARMAQ 001 was cultivated in insect cell medium to an OD_(600 nm)=3.0before the addition of a cryoprotectant and storage at −80° C. Thebacterial content was determined to be 2.6×10⁸ TCID₅₀/ml by end-pointtitration.

An ampoule of frozen isolate PHARMAQ 001 was thawed at room temperatureand a 0.1 ml dose containing 1.3×10⁷ TCID₅₀/ml was injectedintraperitoneally into Atlantic salmon with an average weight of 25-30grams.

The fish were held at 15° C. for 44 days in freshwater. No fish died orshowed any clinical signs of SRS.

Tissue samples (from head kidney and spleen) from vaccinated fish weretaken every week for three weeks after vaccination. The presence ofbacterial genomes in the tissue samples was analyzed by real timequantitative-PCR. The results are shown in Table 2.

TABLE 2 Mean Ct values (9 fish per group) Days post-vaccination headkidney Spleen 7 28.54 28.72 14 30.17 29.42 21 31.69 29.80

As shown in Table 2, the presence of PHARMAQ 001 genomes was generallyfound to peak at 7 days post vaccination with Ct values around 28.

This experiment demonstrates that PHARMAQ 001 is avirulent and does notinduce any symptoms of SRS. Therefore, PHARMAQ 001 is safe and suitablefor use as an attenuated live vaccine.

Example 5: Vaccination with PHARMAQ 001 and Challenge with a Virulent P.salmonis Isolate

To examine the efficacy of the P. salmonis isolate PHARMAQ 001 as avaccine against SRS, Rainbow trout (Oncorhynchus mykiss) with an averageweight of 30 grams were injected with different doses of the vaccineisolate. The vaccine isolate was either preserved as a frozen vaccineand then diluted in PBS for use at a concentration of 2×10⁵ TCID₅₀, oras a lyophilized vaccine and then diluted in PBS for use at aconcentration of 5.2×10⁶ TCID₅₀ per fish.

After an immunization period in fresh water for 504 degree days, thetrout were challenged with a highly virulent wild-type strain of P.salmonis. The virulent wild-type strain was injected intraperitoneallyand the fish were observed for 34 days, as shown in FIG. 1.

FIG. 1 shows the accumulated mortality following challenge with avirulent P. salmonis strain.

The attenuated P. salmonis strain PHARMAQ 001 was found to work well asa live attenuated vaccine, and provided 100% protection against SRS.

In addition, the study shows that PHARMAQ 001 can be either frozen orlyophilized prior to being used as a vaccine.

Example 6: Passage of Attenuated P. salmonis Strain PHARMAQ 001 in FishShows Safety of the Vaccine Strain

To investigate the potential for reversion to virulence, the isolatePHARMAQ 001 was serially passaged through Atlantic salmon. The trial wasperformed in fresh water at 15° C. Atlantic salmon were injected withisolate PHARMAQ 001. Homogenates were prepared from head kidneys 7 daysafter injection, and new Atlantic salmon were injected with thehomogenate.

After a further 7 days, homogenates were prepared from the head kidneysof the second fish, and further Atlantic salmon were injected with thehomogenate.

No mortality or clinical signs of SRS were observed in any fish duringthe trial.

The liver from fish from each passage was homogenized, and homogenateswere tested for the presence of live P. salmonis by plating onto CHABagar plates. Bacteria were only detected after the first passage.

The bacterial loads in liver and spleen were investigated by measuringthe presence of P. salmonis genomes by real time quantitative PCR oneweek after injection of PHARMAQ 001 or homogenate. The results (shown inTable 3) demonstrate that the bacterial loads in spleen and liver werereduced when head kidney homogenates were passaged from the firstinjected fish into passages 2 and 3 of fish.

TABLE 3 Initial injection Passage 2 Passage 3 Liver 28.9 nd nd Spleen28.2 32.8 nd Nd = not detected

This experiment shows that PHARMAQ 001 does not revert to a virulentstrain after serial passage in Atlantic salmon. PHARMAQ 001 is thereforesuitable for use as a live attenuated vaccine.

Example 7: Culturing PHARMAQ 001 in Spinner Flask

Bacterial cultures were grown in ExCell Titer High medium from Sigmawith no supplements. The cultures were incubated in ventilated spinnerflasks at 75 rpm and 20° C., 2 passages after thawing. The growth wasmonitored by OD_(600 nm) measurement.

TABLE 4 Vol. of End Step Media pH Ventilation Stirring Temp InoculumTime OD₆₀₀ 1. ExCell Not Ventilated 75 rpm 20° C. 1% 3 days 3.3 SpinnerTiterHigh controlled cap flask 2. ExCell Not Ventilated 75 rpm 20° C. 1%2 days 4.4 Spinner TiterHigh controlled cap flask

The results shown in Table 4 demonstrate the cultivation of P. salmonisin spinner flasks as the OD_(600 nm) reached 3-4 after 3-4 days ofincubation.

Example 8: Culturing PHARMAQ 0011 in Spinner Flask

P. salmonis strain PHARMAQ 0011 was grown in spinner flasks in Sf-900medium.

PHARMAQ 0011 was harvested from the spinner flask and 20% skimmed milkin water (made from dry skimmed milk and heated for 15 minutes at 80°C.) was added 1:1 to the P. salmonis culture to a final concentration of10% skimmed milk. 2 ml of this mixture (P. salmonis and 10% skimmedmilk) was placed into 10 ml glass vials and then freeze dried in aLabconco FreeZone Triad freeze dryer.

Freeze Drying Cycle:

Freezing: 3 hours hold at −75° C.

Segment 1: ramping rate: 0.1° C./min; holding time: 1 hour; shelftemperature: −40° C.

Segment 2: ramping rate: 0.1° C./min; holding time: 8 hours; shelftemperature: −25° C.

Segment 3: ramping rate: 0.1° C./min; holding time: 24 hours; shelftemperature: −10° C.

Segment 4: ramping rate: 0.1° C./min; holding time: indefinite; shelftemperature: 4° C.

Vacuum was set to 0 μBar in step 1-4

The vials were sealed under vacuum and the process was stopped. Thefreeze-dried materials were stored at 2-8° C.

To determine the viability of the samples, titration (TCID₅₀/ml) wasperformed on the culture at harvest and after freeze drying. Thefreeze-dried cake was rehydrated with 2 ml PBS before titration.

Titer (TCID₅₀/ml) at harvest: 8.7×10⁹ cells/ml

Titer (TCID₅₀/ml) after freeze drying: 3.2×10⁷ cells/ml

Thus, after freeze drying, the samples are sufficiently viable for useas a vaccine.

Example 8: Detecting PHARMAQ 001 Strain

The attenuated live strain of P. salmonis (rpoD/FecR/ATP-grasp domainprotein/FtsH) has been shown to be well tolerated in healthy fish hostsand to colonise the host in a manner consistent with its utility as aneffective vaccine to protect against SRS. It has also been demonstratedto elicit a specific immune response. The PHARMAQ 001 live attenuated P.salmonis strain has been found to be particularly effective for thispurpose.

An assay was developed for identifying and detecting the PHARMAQ 001strain of P. salmonis. The genome of the PHARMAQ 001 strain containssingle nucleotide polymorphisms (SNP) in each of the rpoD, FecR,ATP-grasp domain protein, and FtsH genes, each of which results in achemically significant alteration of the amino acid sequence of theresulting gene product. The assay involves typing each of these singlenucleotide polymorphisms, and determining whether the wild-type ormutant allele is present.

Specifically, the assay involves using target-specific PCR primers toamplify the nucleic acid sequence in the region of the SNP. Separateprobes specific for each of the wild-type and mutant alleles areincluded in the reaction mix. Each probe is labelled with a differentdetectable marker such as a fluorescent dye. In the example describedbelow, the probe specific for the wild-type sequence was labelled withthe FAM fluorophore, and the probe specific for the mutant sequence waslabelled with the VIC fluorescent dye. An overview of the primers andprobes used for each SNP analysis is shown in Table 5. In the presentexample, the SNP analysis was performed using the ABI PRISM® 7900 HTSequence Detection System.

TABLE 5 Target Gene Primer/Probe Sequence Seq. ID No. rpoD FwdGGACAATCCGGATTCCTGTACATAT 67 VProbe1-VIC ACAAGCTTACC T GCGTCTC 68Mutant Allele MProbe2-FAM AAGCTTAAC C GCGTCTC 69 Wild-Type Allele RevGCCGAGTTCTTGGATCATTTGAC 70 ATP-grasp domain Fwd TGGCGATGGTGCGTCTAC 71protein VProbe1-VIC CCATCAG G GAGCCGAG 72 Mutant Allele MProbe2-FAMCCATCAG A GAGCCGAG 73 Wild-Type Allele Rev CGCCAATTGCACCTTGATGAAG 74FtsH Fwd CCAAAAGTTAGGCGGCAAAATTCC 75 VProbe1-VIC TGGGCCAAC T ATCA 76Mutant Allele MProbe2-FAM TGGGCCAAC C ATCA 77 Wild-Type Allele RevGCTAATAGCGTCTTACCTGTTCCA 78

RNA was isolated from cultures of P. salmonis, including PHARMAQ 001 andwild type strains including the virulent starting strain. The sampleswere prepared as shown in Table 6. All tests were performed usingQuantiTect Probe RT-PCR kit (Qiagen).

TABLE 6 Reagent Final concentration 2X Master mix 1x (Recommended by kitsupplier, contains dNTPs, MgCl2 (final concentration 4 mM), HotStartTaqDNA Polymerase, and passive reference dye (ROX) Forward primer 900 nM(0.9 μl of 1.0 μM solution) Reverse Primer 900 nM (0.9 μl of 1.0 μMsolution) VProbe-VIC 200 nM (0.2 μl of 1.0 μM solution) MProbe-FAM 175nM (0.175 μl of 1.0 μM solution) RT-Enzyme mix (QuantiTect)  0.1 μlTemplate 1 pg to 1 μg per reaction dH₂O To 10 μl

Samples were analysed in triplicates on 384-well plates. Each plate wassubjected to a pre-read, for determination of background fluorescence ineach well prior to the real-time RT-PCR step. The real-time RT-PCR wasperformed using standard enzymes and buffers, with the parameters shownin Table 7.

TABLE 7 Step Temperature Time Cycles 1) Reverse transcription 50° C. 30minutes 1 2) DNA polymerase activation 95° C. 15 minutes 1 3)Denaturation, Annealing and 94° C. 15 seconds 45 extension 60° C.  1minute

All primers and probes were optimized to allow annealing and extensionat 60° C. This temperature is also believed to be significant for thecompetition between the two probes in the SNP assay, as it leads tobinding and cleavage of the correct probe as well as destabilization ofthe incorrect probe, depending on the SNP at the probe site.

After the real-time RT-PCR reaction had been performed, the plate wassubjected to an end-point analysis, by performing a post-read of thefluorescence in each well, and comparing the result to the data storedfrom the pre-read. The results are shown in Table 8 (in which a plussign indicates a cycle threshold of less than or equal to 30 and a minussign indicates no detectable fluorescent signal).

TABLE 8 ATP-grasp rpoD domain protein FtsH MProbe2- MProbe2- MProbe2-VProbe1- FAM VProbe1- FAM VProbe1- FAM VIC Wild- VIC Wild- VIC Wild-Mutant Type Mutant Type Mutant Type Allele Allele Allele Allele AlleleAllele PHARMAQ 001, + − + − + − attenuated vaccine strain Virulentstarting strain − + − + − + Wild type P. salmonis − + − + − + strain AWild type P. salmonis − + − + − + strain B Wild type P. salmonis − + − +− + strain C

The assay clearly identified each of the mutant alleles in the PHARMAQ001 strain, and also identified the presence of the wild-type allele inall wild type strains tested. For all tests, the discrimination betweenthe two allelic variants was very good. The assay permits cleardistinction between wild type and PHARMAQ 001 P. salmonis strains.

In order to address various issues and advance the art, the entirety ofthis disclosure shows by way of illustration various embodiments inwhich the claimed invention may be practiced and provide for anattenuated P. salmonis bacterium and an improved P. salmonis vaccine.The advantages and features of the disclosure are of a representativesample of embodiments only, and are not exhaustive and/or exclusive.They are presented only to assist in understanding and teach the claimedfeatures. It is to be understood that advantages, embodiments, examples,functions, features, and/or other aspects of the disclosure are not tobe considered limitations on the disclosure as defined by the claims orlimitations on equivalents to the claims, and that other embodiments maybe utilized and modifications may be made without departing from thescope and/or spirit of the disclosure. Various embodiments may suitablycomprise, consist of, or consist essentially of, various combinations ofthe disclosed elements, components, features, parts, steps, means, etc.In addition, the disclosure includes other inventions not presentlyclaimed, but which may be claimed in future.

1. An attenuated Piscirickettsia salmonis bacterium comprising amutation in the amino acid sequence of each of the rpoD, FecR, ATP-graspdomain protein, and FtsH gene products; wherein the bacterium isavirulent and does not induce symptoms of Salmon Rickettsial Syndromewhen administered to fish.
 2. (canceled)
 3. The attenuated bacterium ofclaim 2 wherein the bacterium does not revert to a virulent strain afterserial passage in fish.
 4. The attenuated bacterium of claim 1 whereinsaid bacterium is capable of inducing immunological protection againstSalmon Rickettsial Syndrome when administered to fish.
 5. The attenuatedbacterium of claim 1 wherein the mutations in the amino acid sequence ineach of the rpoD, FecR, ATP-grasp domain protein, and FtsH genes arenon-reverting mutations.
 6. The attenuated bacterium of claim 1, whereinthe mutations in the amino acid sequence of the rpoD, FecR, ATP-graspdomain protein, and FtsH gene products are mutations corresponding tothe LF-89 wild-type protein, as derived from the LF-89 genomic sequencethat is available under the GenBank accession no. AMFF00000000.2, and asprovided as Seq. ID No.s 17, 26, 40, and 54, respectively.
 7. Theattenuated bacterium of claim 1, comprising a mutation in the region of:a) amino acid residues 462-504 of the rpoD gene product, provided asSeq. ID No. 17; b) amino acid residues 39-137 of the FecR gene product,provided as Seq. ID No. 26; c) amino acid residues 118-251 of theATP-grasp domain protein gene product, provided as Seq. ID No. 40;and/or, d) amino acid residues 152-274 of the FtsH gene product,provided as Seq. ID No.
 54. 8. The attenuated bacterium of claim 1,comprising: a) an arginine to cysteine mutation at position 473 of therpoD gene product, provided as Seq. ID No. 17; b) a premature stop codonat the position corresponding to residue 83 of the FecR gene product,provided as Seq. ID No. 26; c) a serine to proline mutation at position184 of the ATP-grasp domain protein gene product, provided as Seq. IDNo. 40; and/or, d) a methionine to isoleucine mutation at position 191of the FtsH gene product, provided as Seq. ID No.
 54. 9. An attenuatedPiscirickettsia salmonis bacterium comprises the strain PHARMAQ 001deposited with the European Collection of Cell Cultures, Public healthEngland, Culture Collections, Porton Down, Salisbury SP4 OJG, UnitedKingdom, on 9 Oct. 2014 with accession number
 14100901. 10. A live,attenuated vaccine composition comprising: (a) an attenuatedPiscirickettsia salmonis bacterium as claimed in either claim 1 or 9;and (b) a pharmaceutically acceptable carrier or diluent.
 11. A live,attenuated vaccine composition as claimed in claim 10, in freeze-driedform.
 12. A method of producing the attenuated bacterium of either claim1, the method comprising: a) subjecting an initial population of P.salmonis bacteria to attenuating conditions to produce a putativelyattenuated bacterial population; b) identifying clones of the putativelyattenuated bacterial population that have mutations in the amino acidsequences of all of the rpoD, FecR, ATP-grasp domain protein, and FtsHgene products; and, c) identifying and selecting clones that havemutations in the amino acid sequence of all of the rpoD, FecR, ATP-graspdomain protein, and FtsH gene products and that also exhibit reducedvirulence relative to wild-type bacteria of the genus Piscirickettsia.13. A method of raising an immune response in a fish, the methodcomprising administering to the fish the attenuated Piscirickettsiasalmonis bacterium either one of claim 1 or
 9. 14. A method ofvaccinating a fish against Salmon Rickettsial Syndrome, the methodcomprising administering to a fish an immunologically-effective amountof the vaccine composition of claim
 10. 15. (canceled)
 16. (canceled)17. A method of distinguishing between wild-type and mutant alleles of aPiscirickettsia salmonis single nucleotide polymorphism (SNP) located atthe position corresponding to: residue number 1417 of Seq. ID No. 1 (inthe rpoD gene); residue number 247 of Seq. ID No. 4 (in the FecR gene);residue number 550 of Seq. ID No. 7 (in the ATP-grasp domain proteingene); or, residue number 573 of Seq. ID No. 10 (in the FtsH gene),wherein the method comprises: i) amplifying by PCR the region of thenucleotide sequence containing the SNP; ii) including in the PCRreaction mix a nucleic acid probe having a sequence complementary to oneallele of the SNP, the probe comprising a detectable marker; and iii)analysing the PCR product for the presence of the marker, wherein thepresence of the marker is indicative of the presence of the allele. 18.A method as claimed in claim 17, wherein the method further comprisesincluding in the PCR reaction mix a first nucleic acid probe having asequence complementary to the wild-type allele of the SNP, and a secondnucleic acid probe having a sequence complementary to the mutant alleleof the SNP, the first and second probes comprising different detectablemarkers.
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)